TWI840351B - T cell receptors and engineered cells expressing same - Google Patents

Abstract

Provided herein are binding molecules, such as those that recognize or bind a peptide epitope of a cancer antigen, such as expressed on a cancer cell, including cells infected with human papilloma virus (HPV) or that contain HPV DNA sequences and/or those that recognize or bind a peptide epitope of HPV 16 E6 or E7, in the context of a major histocompatibility complex (MHC) molecule. Among the provided binding molecules are T cell receptors (TCRs) or antibodies, including antigen-binding fragments thereof, that bind or recognize such peptide epitopes. The present disclosure further relates to engineered cells comprising such binding molecules, e.g., TCRs or antibodies (and chimeric antigen receptors containing the antibodies), and uses thereof in adoptive cell therapy.

Description

T cell receptor and engineered cells expressing the same

The present invention relates in some aspects to binding molecules, such as binding molecules that recognize or bind to peptide antigenic determinants such as cancer antigens expressed on cancer cells, including cells infected with human papillomavirus (HPV) or containing HPV DNA sequences; and/or binding molecules that recognize or bind to peptide antigenic determinants of HPV 16 E6 or E7 in the context of major histocompatibility complex (MHC) molecules. In particular, the present invention relates to T cell receptors (TCRs) or antibodies that bind or recognize such peptide antigenic determinants, including antigen-binding fragments thereof. The present invention further relates to engineered cells comprising such binding molecules, such as TCRs or antibodies (and chimeric antigen receptors containing such antibodies), and their use in adoptive cell therapy.

Human papillomavirus (HPV) is a common virus in human individuals that can, in some cases, be transmitted by skin-to-skin contact and is a common sexually transmitted virus. Certain subtypes of HPV, such as HPV 16, can cause certain cancers, such as cervical cancer and other cancers. In some cases, cancer can be associated with the expression of HPV oncoproteins E6 and/or E7. For example, HPV E6 and/or E7 can promote cancer progression by targeting tumor suppressor signaling pathways involved in cell growth control. Certain therapeutic agents are available that target HPV 16 expressing cells or cancers, but there is still a need for improved agents against HPV 16. Provided are embodiments that meet such needs.

Provided herein are binding molecules, such as T cell receptors (TCRs), such as recombinant TCRs, that bind to peptide antigenic determinants of HPV 16 E6 or E7 in the context of major histocompatibility complex (MHC) molecules and/or peptide antigenic determinants on cells infected with human papillomavirus (HPV) or containing HPV DNA sequences; engineered cells expressing such molecules, such as engineered immune cells, for example engineered CD4+ and/or CD4+ T cells; and related methods or uses.

Provided herein is a T cell receptor (TCR) or an antigen-binding fragment thereof, comprising an α chain comprising a variable α (Vα) region and a β chain comprising a variable β (Vβ) region, wherein: the Vα region comprises a complementary determining region 1 (CDR-1), a complementary determining region 2 (CDR-2) and a complementary determining region 3 (CDR-3), which comprise the amino acid sequences of SEQ ID NOs: 48, 49 and 50, respectively, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 61, 62 and 63, respectively; the Vα region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 78, 79 and 80, respectively, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 78, 79 and 80, respectively. The Vα region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 89, 90 and 91, respectively; the Vα region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 104, 105 and 106, respectively, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 89, 90 and 115, respectively; the Vα region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 127, 128 and 129, respectively, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 89, 90 and 138, respectively; the Vα region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: NO: 127, 128 and 150, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 89, 90 and 158; the Vα region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 170, 171 and 172, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 181, 182 and 183; the Vα region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 196, 197 and 198, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: NO: 207, 208 and 209; or the Vα region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 222, 223 and 224, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 233, 234 and 235. In a specific embodiment of any of the TCRs provided in this class, the TCR or antigen-binding fragment thereof binds to or recognizes a peptide antigenic determinant of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule. In some embodiments, the peptide antigenic determinant is or comprises E7(11-19)YMLDLQPET (SEQ ID NO: 271).

Also provided herein are TCRs containing a Vα region containing CDR1, CDR2, and CDR3 as contained in a Vα region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 47, 77, 103, 126, 149, 169, 195, or 221, and a Vβ region containing CDR1, CDR2, and CDR3 as contained in a Vβ region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 60, 88, 114, 137, 157, 180, 206, or 232. In specific embodiments of any of the TCRs provided herein, the TCR or antigen-binding fragment thereof binds to or recognizes a peptide antigenic determinant of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule. In some embodiments, the peptide antigen determinant is or comprises E7(11-19)YMLDLQPET (SEQ ID NO: 271).

In some of any such embodiments, the Vα region comprises the amino acid sequence shown in any one of SEQ ID NOs: 47, 77, 103, 126, 149, 169, 195, or 221, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 47, 77, 103, 126, 149, 169, 195, or 221; and the Vβ region comprises the amino acid sequence shown in any one of SEQ ID NOs: 60, 88, 114, 137, 157, 180, 206, or 232, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: No.: Any one of 60, 88, 114, 137, 157, 180, 206 or 232 has an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

Provided herein is a T cell receptor (TCR) or an antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein: the Vα region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 47, 77, 103, 126, 149, 169, 195, or 221, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and/or the Vβ region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 60, 88, 114, 137, 157, 180, 206, or 232, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In specific embodiments of any of the TCRs provided herein, the TCR or antigen-binding fragment thereof binds to or recognizes a peptide antigenic determinant of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule. In some embodiments, the peptide antigenic determinant is or comprises E7(11-19)YMLDLQPET (SEQ ID NO: 271).

Also provided herein is a T cell receptor (TCR) or an antigen-binding fragment thereof, comprising an α chain comprising a variable α (Vα) region and a β chain comprising a variable β (Vβ) region, wherein: the Vα region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 47, 77, 103, 126, 149, 169, 195, or 221, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and the Vβ region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 60, 88, 114, 137, 157, 180, 206, or 232, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In specific embodiments of any of the TCRs provided herein, the TCR or antigen-binding fragment thereof binds to or recognizes a peptide antigenic determinant of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule. In some embodiments, the peptide antigenic determinant is or comprises E7(11-19)YMLDLQPET (SEQ ID NO: 271).

Any of the TCRs provided in this class is a TCR containing CDRs as described herein. In some embodiments, this class of TCRs is a TCR containing a Vα region and a Vβ region, wherein the Vα region contains CDR1, CDR2, and CDR3 contained in a Vα region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 47, 77, 103, 126, 149, 169, 195, or 221, and the Vβ region contains CDR1, CDR2, and CDR3 contained in a Vβ region as shown in any one of SEQ ID NOs: 60, 88, 114, 137, 157, 180, 206, or 232.

In specific embodiments of any of the TCRs provided herein, the Vα region comprises an amino acid sequence as set forth in any of SEQ ID NOs: 47, 149, 169, 195, or 221, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith. In other specific embodiments, the Vα region comprises an amino acid sequence as set forth in any of SEQ ID NOs: 103, 126, or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.

In certain embodiments of any of the TCRs provided herein, Provided herein are T cell receptors (TCRs) or antigen-binding fragments thereof as described herein, wherein: the Vβ region comprises an amino acid sequence as set forth in any of SEQ ID NOs: 60, 180, or 232, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In certain other embodiments, the Vβ region comprises an amino acid sequence as set forth in SEQ ID NO: 206, or an amino acid sequence having at least 90.5%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In certain other embodiments, the Vβ region comprises an amino acid sequence as set forth in SEQ ID NOs: 114 or 137, or an amino acid sequence having at least 97.5%, 98%, or 99% sequence identity thereto. In certain other embodiments, the Vβ region comprises the amino acid sequence shown in SEQ ID NO: 157 or an amino acid sequence having at least 98.5% or 99% sequence identity therewith. In certain other embodiments, the Vβ region comprises the amino acid sequence shown in SEQ ID NO: 88 or an amino acid sequence having at least 99.5% sequence identity therewith.

In specific embodiments of any of the TCRs provided herein, the TCR or antigen-binding fragment thereof binds to or recognizes a peptide antigenic determinant of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule. In some embodiments, the peptide antigenic determinant is or comprises E7(11-19)YMLDLQPET (SEQ ID NO: 271).

In some embodiments, the TCR or antigen-binding fragment thereof, when expressed on the surface of a T cell, stimulates cytotoxic activity against a target cancer cell. In some embodiments, the target cancer cell contains an HPV DNA sequence or expresses HPV 16. In some embodiments, the target cancer cell is SCC152. In some embodiments, the target cancer cell expresses a peptide antigenic determinant of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule. In some embodiments, the peptide antigenic determinant is or comprises E7(11-19)YMLDLQPET (SEQ ID NO: 271).

In a specific embodiment of any of the TCRs provided herein, the Vα region comprises a complementary determining region 1 (CDR-1), CDR-2 and CDR-3, which respectively comprise the CDR-1, CDR-2 and CDR-3 amino acid sequences contained in the Vα region amino acid sequence shown in any one of SEQ ID NOs: 47, 77, 103, 126, 149, 169, 195 or 221; and/or the Vβ region comprises a complementary determining region 1 (CDR-1), CDR-2 and CDR-3, which respectively comprise the CDR-1, CDR-2 and CDR-3 amino acid sequences contained in the Vβ region amino acid sequence shown in any one of SEQ ID NOs: 60, 88, 114, 137, 157, 180, 206 or 232.

In some of any such embodiments: the Vα region comprises a complementation determining region 3 (CDR-3) comprising the amino acid sequence shown in any one of SEQ ID NOs: 50, 80, 106, 129, 150, 172, 198, or 224; and/or the Vβ region comprises a complementation determining region 3 (CDR-3) comprising the amino acid sequence shown in any one of SEQ ID NOs: 63, 91, 115, 138, 158, 183, 209, or 235. In certain embodiments of any of the TCRs provided herein, the Vα region comprises: a complementary determining region 1 (CDR-1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 48, 78, 104, 127, 170, 196, or 222; and/or a complementary determining region 2 (CDR-2) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 49, 79, 105, 128, 171, 197, or 223. In specific embodiments, the Vβ region comprises: a complementary determining region 1 (CDR-1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 61, 89, 181, 207, or 233; and/or a complementary determining region 2 (CDR-2) comprising an amino acid sequence as set forth in SEQ ID NOs: 62, 90, 182, 208, or 234.

In some of any such embodiments: the Vα region comprises CDR-1, CDR-2 and CDR-3 comprising the amino acid sequences of SEQ ID NOs: 48, 49 and 50, respectively, and the Vβ region comprises CDR-1, CDR-2 and CDR-3 comprising the amino acid sequences of SEQ ID NOs: 61, 62 and 63, respectively; the Vα region comprises CDR-1, CDR-2 and CDR-3 comprising the amino acid sequences of SEQ ID NOs: 78, 79 and 80, respectively, and the Vβ region comprises CDR-1, CDR-2 and CDR-3 comprising the amino acid sequences of SEQ ID NOs: 89, 90 and 91, respectively; the Vα region comprises CDR-1, CDR-2 and CDR-3 comprising the amino acid sequences of SEQ ID NOs: 104, 105 and 106, respectively, and the Vβ region comprises CDR-1, CDR-2 and CDR-3 comprising the amino acid sequences of SEQ ID NOs: The Vα region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 89, 90 and 115, respectively; the Vα region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 127, 128 and 129, respectively, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 89, 90 and 138, respectively; the Vα region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 127, 128 and 150, respectively, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 89, 90 and 158, respectively; the Vα region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: NO: 170, 171 and 172, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 181, 182 and 183; the Vα region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 196, 197 and 198, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 207, 208 and 209; or the Vα region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 222, 223 and 224, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 233, 234 and 235.

In certain embodiments, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 47 and 60, respectively, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In certain embodiments, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 77 and 88, respectively, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In certain embodiments, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 77 and 88, respectively, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99.5% sequence identity thereto. In certain embodiments, the Vα region and the Vβ region comprise the amino acid sequence of SEQ ID NOs: 103 and 114, respectively, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, or 99% sequence identity thereto. In certain embodiments, the Vα region and the Vβ region comprise the amino acid sequence of SEQ ID NOs: 126 and 137, respectively, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98%, or 99% sequence identity thereto. In certain embodiments, the Vα region and the Vβ region comprise the amino acid sequence of SEQ ID NOs: 149 and 157, respectively, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98.5%, or 99% sequence identity thereto. In some embodiments, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 169 and 180, respectively, or amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith. In some embodiments, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 195 and 206, respectively, or amino acid sequences having at least 90.5%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith. In some embodiments, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 221 and 232, respectively, or amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith.

In a specific embodiment: the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 47 and 60, respectively. In a specific embodiment, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 77 and 88, respectively. In a specific embodiment, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 77 and 88, respectively. In a specific embodiment, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 103 and 114, respectively. In a specific embodiment, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 126 and 137, respectively. In a specific embodiment, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 149 and 157, respectively. In a specific embodiment, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 169 and 180, respectively. In a specific embodiment, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 195 and 206, respectively. In a specific embodiment: the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 221 and 232, respectively.

In some of any of the embodiments, the Vα region comprises a complementary determining region 1 (CDR-1), CDR-2 and CDR-3, which respectively comprise the CDR-1, CDR-2 and CDR-3 amino acid sequences contained in the Vα region amino acid sequence shown in SEQ ID NO: 47; and the Vβ region comprises a complementary determining region 1 (CDR-1), CDR-2 and CDR-3, which respectively comprise the CDR-1, CDR-2 and CDR-3 amino acid sequences contained in the Vβ region amino acid sequence shown in SEQ ID NO: 60.

In some of any such embodiments: the Vα region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 48, 49 and 50, respectively, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 61, 62 and 63. In some of any such embodiments, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 47 and 60, respectively.

In certain embodiments: the Vα region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 196, 197 and 198, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 207, 208 and 209. In some of any such embodiments, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 195 and 206, respectively.

In certain embodiments, the Vα region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 222, 223 and 224, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 233, 234 and 235. In some of any such embodiments, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 221 and 232, respectively.

In some of any such embodiments, the α chain further comprises an α homeostatic (Cα) region and/or the β chain further comprises a β homeostatic (Cβ) region. In certain embodiments, the Cα region and the Cβ region are mouse homeostatic regions. In specific embodiments, the Cα region and the Cβ region are human homeostatic regions. In specific embodiments, the Cα region and/or the Cβ region comprise the introduction of one or more cysteine residues capable of forming one or more non-native disulfide bridges between the α chain and the β chain.

In some of any such embodiments, the Cα region comprises the amino acid sequence shown in SEQ ID NO: 15, 275, 276, 277, 278, 279, 280, 281 or 282, or an amino acid sequence having at least 90% sequence identity to the sequence shown in SEQ ID NO: 15, 275, 276, 277, 278, 279, 280, 281 or 282; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 30, 283, 284 or 285, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 30, 283, 284 or 285. In certain embodiments, the Cα region comprises the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 90% sequence identity therewith.

In specific embodiments: the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 44, 74, 100, 123, 146, 166, 192 or 218, or an amino acid sequence having at least 90% sequence identity thereto; and/or the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 57, 85, 111, 134, 154, 177, 203 or 229, or an amino acid sequence having at least 90% sequence identity thereto. In some embodiments, the alpha chain and the beta chain comprise the amino acid sequences of SEQ ID NO: 44 and 57, respectively. In some embodiments, the alpha chain and the beta chain comprise the amino acid sequences of SEQ ID NO: 74 and 85, respectively. In some embodiments, the alpha chain and the beta chain comprise the amino acid sequences of SEQ ID NO: 100 and 111, respectively. In some embodiments, the α chain and the β chain comprise the amino acid sequences of SEQ ID NOs: 123 and 134, respectively. In some embodiments, the α chain and the β chain comprise the amino acid sequences of SEQ ID NOs: 146 and 154, respectively. In some embodiments, the α chain and the β chain comprise the amino acid sequences of SEQ ID NOs: 166 and 177, respectively. In some embodiments, the α chain and the β chain comprise the amino acid sequences of SEQ ID NOs: 192 and 202, respectively. In some embodiments, the α chain and the β chain comprise the amino acid sequences of SEQ ID NOs: 218 and 229, respectively.

In some of any such embodiments, the Cα region includes an amino acid sequence shown in SEQ ID NO: 14, 333, 334, 335, 336, 337, 338, 341, 344, 345, 346, 347 or 348, or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region includes an amino acid sequence shown in SEQ ID NO: 29, 339, 340, 342, 343, 350, 351 or 352, or an amino acid sequence having at least 90% sequence identity therewith.

In some of any such embodiments, the Cα region comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 90% sequence identity therewith. In some of any such embodiments, the Cα region comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 350 or an amino acid sequence having at least 90% sequence identity therewith.

In some of any such embodiments, the introduction of one or more cysteine residues comprises replacing a non-cysteine residue with a cysteine residue. In some of any such embodiments, the provided TCR or antigen-binding fragment thereof comprises a Cα region comprising a cysteine at a position corresponding to position 48 as shown in any one of SEQ ID NOs: 333-337 or at a position corresponding to position 49 as shown in SEQ ID NOs: 338 or 341; and/or a Cβ region comprising a cysteine at a position corresponding to position 57 as shown in SEQ ID NOs: 339 or 340 or at a position corresponding to position 58 as shown in SEQ ID NOs: 342 or 343.

In certain embodiments: the alpha chain comprises an amino acid sequence as set forth in SEQ ID NO: 43, 73, 99, 122, 145, 165, 191 or 217, or an amino acid sequence having at least 90% sequence identity thereto; and/or the beta chain comprises an amino acid sequence as set forth in SEQ ID NO: 56, 84, 110, 133, 153, 176, 202 or 228, or an amino acid sequence having at least 90% sequence identity thereto. In certain embodiments, the alpha chain and the beta chain comprise the amino acid sequences of SEQ ID NO: 43 and 56, respectively. In certain embodiments, the alpha chain and the beta chain comprise the amino acid sequences of SEQ ID NO: 73 and 84, respectively. In certain embodiments, the alpha chain and the beta chain comprise the amino acid sequences of SEQ ID NO: 99 and 110, respectively. In some embodiments, the α chain and the β chain comprise the amino acid sequences of SEQ ID NOs: 122 and 133, respectively. In some embodiments, the α chain and the β chain comprise the amino acid sequences of SEQ ID NOs: 145 and 153, respectively. In some embodiments, the α chain and the β chain comprise the amino acid sequences of SEQ ID NOs: 165 and 176, respectively. In some embodiments, the α chain and the β chain comprise the amino acid sequences of SEQ ID NOs: 191 and 201, respectively. In some embodiments, the α chain and the β chain comprise the amino acid sequences of SEQ ID NOs: 217 and 228, respectively.

In some of any such embodiments, the alpha chain comprises the amino acid sequence shown in SEQ ID NO: 43, 73, 99, 122, 145, 165, 191 or 217, or an amino acid sequence having at least 90% sequence identity thereto; and/or the beta chain comprises the amino acid sequence shown in SEQ ID NO: 359, 370, 371, 372, 373, 374, 375 or 376, or an amino acid sequence having at least 90% sequence identity thereto. In some embodiments, the alpha chain and the beta chain comprise the amino acid sequences of SEQ ID NO: 43 and 359, respectively. In some embodiments, the alpha chain and the beta chain comprise the amino acid sequences of SEQ ID NO: 73 and 370, respectively. In some embodiments, the alpha chain and the beta chain comprise the amino acid sequences of SEQ ID NO: 99 and 371, respectively. In some embodiments, the α chain and the β chain comprise the amino acid sequences of SEQ ID NOs: 122 and 372, respectively. In some embodiments, the α chain and the β chain comprise the amino acid sequences of SEQ ID NOs: 145 and 373, respectively. In some embodiments, the α chain and the β chain comprise the amino acid sequences of SEQ ID NOs: 165 and 374, respectively. In some embodiments, the α chain and the β chain comprise the amino acid sequences of SEQ ID NOs: 191 and 375, respectively. In some embodiments, the α chain and the β chain comprise the amino acid sequences of SEQ ID NOs: 217 and 376, respectively.

In certain embodiments, the TCR or antigen-binding fragment comprises: (i) an alpha chain and a beta chain comprising the amino acid sequences of SEQ ID NOs: 44 and 57, respectively; (ii) an alpha chain and a beta chain comprising the amino acid sequences shown in SEQ ID NOs: 43 and 56, respectively; (iii) an alpha chain and a beta chain comprising the amino acid sequences shown in SEQ ID NOs: 43 and 359, respectively; or (iv) an alpha chain and a beta chain independently exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the alpha chain and the beta chain sequences in (i), respectively, or to the alpha chain and the beta chain sequences in (ii), respectively, wherein the alpha chain comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 43 and 359. NO: amino acid sequences of 48, 49 and 50, and the β chain includes CDR-1, CDR-2 and CDR-3, which include amino acid sequences of SEQ ID NO: 61, 62 and 63.

In some of any such embodiments, the TCR or antigen-binding fragment comprises an alpha chain and a beta chain independently exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to an alpha chain and a beta chain sequence comprising the amino acid sequences of SEQ ID NOs: 43 and 56, respectively, wherein the alpha chain comprises CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NOs: 48, 49, and 50, respectively, and the beta chain comprises CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences of SEQ ID NOs: 61, 62, and 63. In some of any such embodiments, the TCR or antigen-binding fragment comprises an alpha chain and a beta chain comprising the amino acid sequences of SEQ ID NOs: 43 and 56, respectively. In some of any such embodiments, the TCR or antigen-binding fragment comprises an alpha chain and a beta chain comprising the amino acid sequences of SEQ ID NOs: 43 and 56, respectively.

In some of any such embodiments, the TCR or antigen-binding fragment comprises an alpha chain and a beta chain independently exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to an alpha chain and a beta chain sequence comprising the amino acid sequences of SEQ ID NOs: 43 and 359, respectively, wherein the alpha chain comprises CDR-1, CDR-2 and CDR-3 comprising the amino acid sequences of SEQ ID NOs: 48, 49 and 50, respectively, and the beta chain comprises CDR-1, CDR-2 and CDR-3 comprising the amino acid sequences of SEQ ID NOs: 61, 62 and 63. In some of any such embodiments, the TCR or antigen-binding fragment comprises an alpha chain and a beta chain comprising the amino acid sequences of SEQ ID NOs: 43 and 359, respectively.

In some of any such embodiments, the TCR or antigen-binding fragment comprises: (i) an alpha chain and a beta chain comprising the amino acid sequences of SEQ ID NOs: 192 and 203, respectively; (ii) an alpha chain and a beta chain comprising the amino acid sequences shown in SEQ ID NOs: 191 and 202, respectively; (iii) an alpha chain and a beta chain comprising the amino acid sequences of SEQ ID NOs: 191 and 375; or (iv) an alpha chain and a beta chain independently exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the alpha chain and the beta chain sequences in (i), respectively, or to the alpha chain and the beta chain sequences in (ii), respectively, wherein the alpha chain comprises CDR-1, CDR-2, and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 191 and 202, respectively; (iv) an alpha chain and a beta chain independently exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the alpha chain and the beta chain sequences in (i), respectively, or to the alpha chain and the beta chain sequences in (ii), respectively, NO: 196, 197 and 198, and the β chain comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 207, 208 and 209. In some of any such embodiments, the TCR or antigen-binding fragment comprises an α chain and a β chain containing the amino acid sequences shown in SEQ ID NO: 191 and SEQ ID NO: 375.

In certain embodiments, the TCR or antigen-binding fragment comprises: (i) an alpha chain and a beta chain comprising the amino acid sequences of SEQ ID NOs: 218 and 229, respectively; (ii) an alpha chain and a beta chain comprising the amino acid sequences shown in SEQ ID NOs: 217 and 228, respectively; (iii) an alpha chain and a beta chain comprising the amino acid sequences of SEQ ID NOs: 217 and 376, respectively; (iv) an alpha chain and a beta chain independently exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to the alpha chain and the beta chain sequences in (i), respectively, or to the alpha chain and the beta chain sequences in (ii), respectively, wherein the alpha chain comprises CDR-1, CDR-2, and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: NO: 222, 223 and 224, and the β chain comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 233, 234 and 235. In some of any such embodiments, the TCR or antigen-binding fragment comprises an α chain and a β chain containing the amino acid sequences of SEQ ID NO: 217 and 376, respectively.

Provided herein is a T cell receptor (TCR) or an antigen-binding fragment thereof, comprising an α chain comprising a variable α (Vα) region and a β chain comprising a variable β (Vβ) region, wherein: the Vα region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 248; a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 249; and a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 250; and the Vβ region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 259; a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 260; and a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: NO: 261. In some of any such embodiments, the Vα region comprises the amino acid sequence shown in any one of SEQ ID NO: 247 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and/or the Vβ region comprises the amino acid sequence shown in any one of SEQ ID NO: 258 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some of any such embodiments, the TCR or antigen-binding fragment thereof binds to or recognizes a peptide antigenic determinant of human papillomavirus (HPV) 16 E6 in the context of an MHC molecule, the peptide antigenic determinant being or comprising E6 (29-38) TIHDIILECV (SEQ ID NO: 268).

Provided herein is a T cell receptor (TCR) or an antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein: the Vα region comprises the amino acid sequence shown in any one of SEQ ID NO: 247, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and/or the Vβ region comprises the amino acid sequence shown in any one of SEQ ID NO: 258, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some of any such embodiments, the TCR or antigen-binding fragment thereof binds to or recognizes a peptide antigenic determinant of human papillomavirus (HPV) 16 E6 in the context of an MHC molecule, the peptide antigenic determinant being or comprising E6(29-38)TIHDIILECV (SEQ ID NO: 268).

Provided herein is a T cell receptor (TCR) or an antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein: the Vα region comprises the amino acid sequence set forth in any one of SEQ ID NO: 247, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and the Vβ region comprises the amino acid sequence set forth in any one of SEQ ID NO: 258, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some of any such embodiments, the TCR or antigen-binding fragment thereof binds to or recognizes a peptide antigenic determinant of human papillomavirus (HPV) 16 E6 in the context of an MHC molecule, the peptide antigenic determinant being or comprising E6(29-38)TIHDIILECV (SEQ ID NO: 268).

In certain embodiments: the Vα region comprises an amino acid sequence as set forth in any one of SEQ ID NO: 247 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and/or the Vβ region comprises an amino acid sequence as set forth in any one of SEQ ID NO: 258 or an amino acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some of any such embodiments, the TCR or antigen-binding fragment thereof binds to or recognizes a peptide antigenic determinant of human papillomavirus (HPV) 16 E6 in the context of an MHC molecule, the peptide antigenic determinant being or comprising E6(29-38)TIHDIILECV (SEQ ID NO: 268).

In certain embodiments: the Vα region comprises a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 250 or a CDR3 contained in the amino acid sequence shown in SEQ ID NO: 247; and/or the Vβ region comprises a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 261 or a CDR3 contained in the amino acid sequence shown in SEQ ID NO: 258. In specific embodiments, the Vα region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 248 or a CDR-1 contained in the amino acid sequence shown in SEQ ID NO: 247; and/or a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 249 or a CDR-2 contained in the amino acid sequence shown in SEQ ID NO: 247. In some of any such embodiments, the Vβ region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 259 or a CDR-1 contained in the amino acid sequence shown in SEQ ID NO: 258; and/or a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 260 or a CDR-2 contained in the amino acid sequence shown in SEQ ID NO: 258.

In certain embodiments: the Vα region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 248; a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 249; and/or a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 250; and/or the Vβ region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 259; a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 260; and/or a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 261.

In a specific embodiment: the Vα region comprises a complementary determining region 1 (CDR-1), CDR-2 and CDR-3, which respectively comprise the CDR-1, CDR-2 and CDR-3 amino acid sequences contained in the Vα region amino acid sequence shown in SEQ ID NO: 247; and/or the Vβ region comprises a complementary determining region 1 (CDR-1), CDR-2 and CDR-3, which respectively comprise the CDR-1, CDR-2 and CDR-3 amino acid sequences contained in the Vβ region amino acid sequence shown in SEQ ID NO: 258. In some of any such embodiments: the Vα region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 248, 249 and 250; and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 259, 260 and 261. In certain embodiments, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 247 and 258, respectively, or amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith. In a specific embodiment: the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 247 and 258, respectively.

In some of any such embodiments, the α chain further comprises an α homeostasis (Cα) region and/or the β chain further comprises a β homeostasis (Cβ) region. In certain embodiments, the Cα region and the Cβ region are mouse homeostasis regions. In specific embodiments, the Cα region and the Cβ region are human homeostasis regions. In some of any such embodiments, the Cα region and/or the Cβ region comprises the introduction of one or more cysteine residues capable of forming one or more non-native disulfide bridges between the α chain and the β chain.

In some of any such embodiments, the Cα region includes the amino acid sequence shown in SEQ ID NO: 15, 275, 276, 277, 278, 279, 280, 281 or 282, or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region includes the amino acid sequence shown in SEQ ID NO: 30, 283, 284 or 285, or an amino acid sequence having at least 90% sequence identity therewith. In certain embodiments: the Cα region includes the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region includes the amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence having at least 90% sequence identity therewith.

In specific embodiments: a) the alpha chain comprises the amino acid sequence shown in SEQ ID NO: 244 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and/or the beta chain comprises the amino acid sequence shown in SEQ ID NO: 255 or an amino acid sequence having at least 90% sequence identity thereto. In some embodiments, the alpha chain comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 248, 249 and 250, and the beta chain comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 259, 260 and 261; or b) the alpha chain and the beta chain comprise the amino acid sequences of SEQ ID NO: 244 and 255, respectively.

In some of any such embodiments, the Cα region includes an amino acid sequence shown in SEQ ID NO: 14, 333, 334, 335, 336, 337, 338, 341, 344, 345, 346, 347 or 348, or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region includes an amino acid sequence shown in SEQ ID NO: 29, 339, 340, 342, 343, 350, 351 or 352, or an amino acid sequence having at least 90% sequence identity therewith.

In some of any such embodiments: the Cα region comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 90% sequence identity therewith. In some of any such embodiments, the Cα region comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 350 or an amino acid sequence having at least 90% sequence identity therewith.

In some of any such embodiments, the introduction of one or more cysteine residues comprises replacing a non-cysteine residue with a cysteine residue. In some of any such embodiments, the Cα region comprises a cysteine at a position corresponding to position 48 as shown in any one of SEQ ID NOs: 333-337 or at a position corresponding to position 49 as shown in SEQ ID NOs: 338 or 341; and/or the Cβ region comprises a cysteine at a position corresponding to position 57 as shown in SEQ ID NOs: 339 or 340 or at a position corresponding to position 58 as shown in SEQ ID NOs: 342 or 343.

In certain embodiments: a) the α chain comprises the amino acid sequence shown in SEQ ID NO: 243 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and/or the β chain comprises the amino acid sequence shown in SEQ ID NO: 254 or an amino acid sequence having at least 90% sequence identity thereto. In some embodiments, the α chain comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 248, 249 and 250, and the β chain comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 259, 260 and 261; or b) the α chain and the β chain comprise the amino acid sequences of SEQ ID NO: 243 and 254, respectively.

In some of any such embodiments, the alpha chain comprises the amino acid sequence shown in SEQ ID NO: 243 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and/or the beta chain comprises the amino acid sequence shown in SEQ ID NO: 377 or an amino acid sequence having at least 90% sequence identity thereto. In some embodiments, the alpha chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 248, 249 and 250, respectively, and the beta chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 259, 260 and 261, respectively. In some embodiments, the alpha chain and the beta chain comprise the amino acid sequences of SEQ ID NOs: 243 and 377, respectively.

Provided herein is a T cell receptor (TCR) or an antigen-binding fragment thereof, comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein: the Vα region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 10; a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 11; and/or a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 12; and/or the Vβ region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 25; a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 26; and/or a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: NO: The amino acid sequence shown in 27. In certain embodiments, the TCR or its antigen-binding fragment, when expressed on the surface of a T cell, stimulates cytotoxic activity against a target cancer cell. In certain embodiments, the target cancer cell contains an HPV DNA sequence or expresses HPV 16 and/or, as the case may be, the target cancer cell is SCC152.

In some of any such embodiments, the Vα region comprises the amino acid sequence shown in SEQ ID NO: 9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and the Vβ region comprises the amino acid sequence shown in SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

Provided herein are T cell receptors (TCRs) or antigen-binding fragments thereof comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein: the Vα region comprises an amino acid sequence as set forth in any one of SEQ ID NO: 9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and/or the Vβ region comprises an amino acid sequence as set forth in any one of SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In certain embodiments, the TCR or antigen-binding fragment thereof, when expressed on the surface of a T cell, stimulates cytotoxic activity against target cancer cells. In some embodiments, the target cancer cell contains an HPV DNA sequence or expresses HPV 16 and/or optionally the target cancer cell is SCC152.

Provided herein are T cell receptors (TCRs) or antigen-binding fragments thereof comprising an alpha chain comprising a variable alpha (Vα) region and a beta chain comprising a variable beta (Vβ) region, wherein: the Vα region comprises an amino acid sequence as set forth in any one of SEQ ID NO: 9, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and the Vβ region comprises an amino acid sequence as set forth in any one of SEQ ID NO: 24, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In certain embodiments, the TCR or antigen-binding fragment thereof, when expressed on the surface of a T cell, stimulates cytotoxic activity against target cancer cells. In some embodiments, the target cancer cell contains an HPV DNA sequence or expresses HPV 16 and/or optionally the target cancer cell is SCC152.

In some of any such embodiments: the Vα region comprises an amino acid sequence as set forth in any one of SEQ ID NO: 9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and/or the Vβ region comprises an amino acid sequence as set forth in any one of SEQ ID NO: 24 or an amino acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In certain embodiments, the TCR or antigen-binding fragment thereof, when expressed on the surface of a T cell, stimulates cytotoxic activity against a target cancer cell. In some embodiments, the target cancer cell contains an HPV DNA sequence or expresses HPV 16 and/or optionally wherein the target cancer cell is SCC152.

In specific embodiments: the Vα region comprises a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 12 or a CDR3 contained within the amino acid sequence shown in SEQ ID NO: 9; and/or the Vβ region comprises a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 27 or a CDR3 contained within the amino acid sequence shown in SEQ ID NO: 24. In some of any such embodiments, the Vα region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 10 or a CDR-1 contained within the amino acid sequence shown in SEQ ID NO: 9; and/or a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 11 or a CDR-2 contained within the amino acid sequence shown in SEQ ID NO: 9. In certain embodiments, the Vβ region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 25 or a CDR-1 contained in the amino acid sequence shown in SEQ ID NO: 24; and/or a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 26 or a CDR-2 contained in the amino acid sequence shown in SEQ ID NO: 24.

In a specific embodiment: the Vα region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 10; a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 11; and/or a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 12; and/or the Vβ region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 25; a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 26; and/or a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 27.

In some of any such embodiments: the Vα region comprises a complementary determining region 1 (CDR-1), CDR-2 and CDR-3, which respectively comprise the CDR-1, CDR-2 and CDR-3 amino acid sequences contained in the Vα region amino acid sequence shown in SEQ ID NO: 9; and/or the Vβ region comprises a complementary determining region 1 (CDR-1), CDR-2 and CDR-3, which respectively comprise the CDR-1, CDR-2 and CDR-3 amino acid sequences contained in the Vβ region amino acid sequence shown in SEQ ID NO: 24. In certain embodiments: the Vα region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 10, 11 and 12, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 25, 26 and 27. In certain embodiments, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 9 and 24, respectively, or amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith. In some of any such embodiments, the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 9 and 24, respectively.

In certain embodiments, the α chain further comprises an α constant region (Cα) and/or the β chain further comprises a β constant region (Cβ). In certain embodiments, the Cα region and the Cβ region are mouse constant regions. In some of any such embodiments, the Cα region and the Cβ region are human constant regions. In certain embodiments, the Cα region and/or the Cβ region comprise the introduction of one or more cysteine residues capable of forming one or more non-natural disulfide bridges between the α chain and the β chain. In some of any such embodiments, the Cα region includes the amino acid sequence shown in SEQ ID NO: 15, 275, 276, 277, 278, 279, 280, 281 or 282, or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region includes the amino acid sequence shown in SEQ ID NO: 30, 283, 284 or 285, or an amino acid sequence having at least 90% sequence identity therewith.

In a specific embodiment: the Cα region comprises the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 90% sequence identity therewith.

In some of any such embodiments: a) the alpha chain comprises the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and/or the beta chain comprises the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 90% sequence identity thereto. In some embodiments, the alpha chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, and the beta chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 25, 26 and 27, respectively; or b) the alpha chain and the beta chain comprise the amino acid sequences of SEQ ID NOs: 6 and 21, respectively.

In some of any such embodiments, the Cα region includes an amino acid sequence shown in SEQ ID NO: 14, 333, 334, 335, 336, 337, 338, 341, 344, 345, 346, 347 or 348, or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region includes an amino acid sequence shown in SEQ ID NO: 29, 339, 340, 342, 343, 350, 351 or 352, or an amino acid sequence having at least 90% sequence identity therewith.

In certain embodiments: the Cα region comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 90% sequence identity therewith. In some of any such embodiments, the Cα region comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 350 or an amino acid sequence having at least 90% sequence identity therewith.

In some of any such embodiments, the introduction of one or more cysteine residues comprises replacing a non-cysteine residue with a cysteine residue. In some of any such embodiments, the Cα region comprises a cysteine at a position corresponding to position 48 as shown in any one of SEQ ID NOs: 333-337 or at a position corresponding to position 49 as shown in SEQ ID NOs: 338 or 341; and/or the Cβ region comprises a cysteine at a position corresponding to position 57 as shown in SEQ ID NOs: 339 or 340 or at a position corresponding to position 58 as shown in SEQ ID NOs: 342 or 343.

In specific embodiments: a) the alpha chain comprises the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and/or the beta chain comprises the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence having at least 90% sequence identity thereto. In some embodiments, the alpha chain comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 10, 11 and 12, and the beta chain comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 25, 26 and 27; or b) the alpha chain and the beta chain comprise the amino acid sequences of SEQ ID NO: 5 and 20, respectively.

In some of any such embodiments, a) the alpha chain comprises the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and/or the beta chain comprises the amino acid sequence shown in SEQ ID NO: 369 or an amino acid sequence having at least 90% sequence identity thereto, whereby the alpha chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NO: 10, 11 and 12, respectively, and the beta chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NO: 25, 26 and 27, respectively; or b) the alpha chain and the beta chain comprise the amino acid sequences of SEQ ID NO: 5 and 369, respectively.

In some of any such embodiments, the TCR or its antigen-binding fragment is encoded by a nucleotide sequence that has been codon-optimized. In some embodiments, the alpha chain and/or the beta chain further comprises a signal peptide. In specific embodiments, the TCR or its antigen-binding fragment is isolated or purified or recombinant. In some of any such embodiments, the TCR or its antigen-binding fragment is recombinant. In some of any such embodiments, the TCR or its antigen-binding fragment is human. In some embodiments, the TCR or its antigen-binding fragment is monoclonal. In specific embodiments, the TCR or its antigen-binding fragment is a single chain. In some of any such embodiments, the TCR or its antigen-binding fragment comprises two chains.

In certain embodiments, the antigen specificity is at least partially CD8-independent.

In certain embodiments, the MHC molecule is an HLA-A2 molecule.

Provided herein is a nucleic acid molecule encoding a provided TCR or antigen-binding fragment thereof or any of its alpha or beta chains. In some of any such embodiments, the nucleotide sequence is codon-optimized. In certain embodiments, the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a peptide sequence that causes ribosome skipping. In some of any such embodiments, the peptide that causes ribosome skipping is a P2A or T2A peptide and/or comprises an amino acid sequence shown in SEQ ID NO: 32 or 302. In specific embodiments, the nucleic acid is synthetic. In some of any such embodiments, the nucleic acid is cDNA.

Provided herein is a vector comprising any of the nucleic acids provided herein. In certain embodiments, the vector is an expression vector. In certain embodiments, the vector is a viral vector. In certain of any such embodiments, the viral vector is a retroviral vector. In certain embodiments, the viral vector is a lentiviral vector. In certain embodiments, the lentiviral vector is derived from HIV-1.

Provided herein is an engineered cell comprising any nucleic acid molecule provided herein or any vector provided herein. Also provided herein is an engineered cell comprising any TCR or antigen binding fragment thereof provided herein. In some of any such embodiments, the TCR or antigen binding fragment thereof is heterologous to the cell. In certain embodiments, the engineered cell is a cell strain. In a specific embodiment, the engineered cell is a primary cell obtained from an individual. In some of any such embodiments, the individual is a mammalian individual. In certain embodiments, the individual is a human. In a specific embodiment, the engineered cell is a T cell. In some of any such embodiments, the T cell is CD8+. In certain embodiments, the T cell is CD4+. In some of any such embodiments, the cell is a human cell.

In some of any such embodiments, the TCR or antigen-binding fragment thereof comprises a human α constitutive (Cα) region and a human β constitutive (Cβ) region and the engineered cell comprises or expresses an endogenous human TCR. In some of any such embodiments, the Vα region comprises CDR-1, CDR-2, and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 48, 49, and 50, respectively, and the Vβ region comprises CDR-1, CDR-2, and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 61, 62, and 63. In some of any such embodiments, the Vα region of the TCR or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 47, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and the Vβ region comprises the amino acid sequence set forth in SEQ ID NO: 60, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any such embodiments, the Vα region and the Vβ region of the TCR or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs: 47 and 60, respectively. In some of any such embodiments, the Cα region of the TCR or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity thereto; and/or the Cβ region of the TCR or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 350 or an amino acid sequence having at least 90% sequence identity thereto.

Provided herein are engineered cells containing a recombinant human TCR containing a human α constant (Cα) region and a human β constant (Cβ) region, and the engineered cells include or express endogenous human TCRs. In some of any such embodiments, the recombinant human TCR includes a Vα region containing CDR-1, CDR-2, and CDR-3, which include the amino acid sequences of SEQ ID NOs: 48, 49, and 50, respectively; and a Vβ region containing CDR-1, CDR-2, and CDR-3, which include the amino acid sequences of SEQ ID NOs: 61, 62, and 63. In some of any such embodiments, the Vα region of the TCR or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 47, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and the Vβ region comprises the amino acid sequence set forth in SEQ ID NO: 60, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any such embodiments, the Vα region and the Vβ region of the TCR or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs: 47 and 60, respectively. In some of any such embodiments, the Cα region of the TCR or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity thereto; and/or the Cβ region of the TCR or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 350 or an amino acid sequence having at least 90% sequence identity thereto.

In certain embodiments, the engineered cells comprise a genetic disruption of a T cell receptor alpha homeostatic ( TRAC ) gene and/or a T cell receptor beta homeostatic ( TRBC ) gene. In some of any such embodiments, the TRBC gene is one or both of a T cell receptor beta homeostatic 1 ( TRBC1 ) or a T cell receptor beta homeostatic 2 ( TRBC2 ) gene.

Provided herein is a method for producing a cell of any of the provided embodiments, the method comprising introducing any of the provided vectors into the cell in vitro or ex vivo. In certain embodiments, the vector is a viral vector and the introduction is performed by transduction. In a specific embodiment, the method further comprises introducing one or more agents into the cell, wherein each of the one or more agents is independently capable of inducing gene disruption of the T cell receptor alpha constant ( TRAC ) gene and/or the T cell receptor beta constant ( TRBC ) gene. In some of any such embodiments, the one or more agents capable of inducing gene disruption comprise a DNA binding protein or a DNA binding nucleic acid that specifically binds or hybridizes to a target site.

In certain embodiments, the one or more agents capable of inducing gene disruption comprise (a) a fusion protein comprising a DNA targeting protein and a nuclease or (b) an RNA-guided nuclease. In particular embodiments, the DNA targeting protein or RNA -guided nuclease comprises a zinc finger protein (ZFP), a TAL protein, or a clustered regularly interspaced short palindromic nucleic acid (CRISPR)-associated nuclease (Cas) specific for a target site within the TRAC and/or TRBC gene. In some of any such embodiments, the one or more agents comprise a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas9 combination that specifically binds to, recognizes, or hybridizes with a target site. In certain embodiments, each of the one or more agents comprises a guide RNA (gRNA) having a targeting domain complementary to at least one target site. In specific embodiments, the one or more agents are introduced as a ribonucleoprotein (RNP) complex comprising a gRNA and a Cas9 protein. In some of any such embodiments, the RNP is introduced by electroporation, a particle gun, calcium phosphate transfection, cell compression or extrusion. In certain embodiments, the RNP is introduced by electroporation. In specific embodiments, the one or more agents are introduced as one or more polynucleotides encoding gRNA and/or Cas9 protein.

Provided herein is a composition comprising any of the engineered cells described herein. In some of any such embodiments, the engineered cells comprise CD4+ and/or CD8+ T cells. In certain embodiments, the engineered cells comprise CD4+ and CD8+ T cells. Also provided herein is a composition comprising the engineered CD8+ cells described herein and the engineered CD4+ cells described herein. In specific embodiments, the TCR or antigen-binding fragment thereof binds to or recognizes a peptide antigenic determinant of HPV 16 in the context of an MHC molecule, at least in part, CD8-independently.

In some of any such embodiments, the CD8+ cells and CD4+ cells are engineered with the same TCR or antigen binding fragment thereof and/or each is engineered with a TCR or antigen binding fragment thereof that binds to or recognizes the same peptide antigenic determinant of HPV 16 in the context of an MHC molecule. In certain embodiments, the composition further comprises a pharmaceutically acceptable excipient.

Provided herein is a method of treatment comprising administering any of the provided engineered cells to an individual suffering from a disease or condition associated with HPV. Also provided herein is a method of treatment comprising administering any of the provided compositions to an individual suffering from a disease or condition associated with HPV. In a specific embodiment, the disease or condition is associated with HPV16. In a specific embodiment, the disease or condition is cancer.

Provided herein is a method of treatment comprising administering an engineered cell described herein to an individual suffering from a disease or condition associated with HPV. Also provided herein is a method of treatment comprising administering a composition described herein to an individual suffering from cancer. Provided herein is any one of the compositions described herein for use in treating a disease or condition associated with HPV in an individual.

Provided herein is a use of a composition for the manufacture of a medicament for treating a disease or condition associated with HPV in an individual. In some of any such embodiments, the disease or condition is associated with HPV16. In particular embodiments, the disease or condition is cancer. Also provided herein is a composition described herein for treating cancer in an individual. Also provided herein is a use of any of the compositions described herein for the manufacture of a medicament for treating cancer in an individual. In particular embodiments, the individual is a human.

Figure 1 shows the lytic activity of TCR-transduced primary T cells cultured with SCC152 target cells based on the loss of viable target cells at various time points assessed.

Figure 2 shows the lytic activity of TCR-transduced naive T cells expressing TCR 57 cultured with SCC152 target cells at various effector:target (E:T) ratios compared to controls at various time points assessed, based on the loss of live target cells.

FIG. 3 shows the results of an assessment of the ability of reference TCR-transduced primary human T cells to lyse target cells in an antigen-specific manner using cancer cell lines expressing the antigen.

FIG. 4A shows cytolytic activity and interleukin production of CD8+ T cells expressing TCR, as measured by culturing effector cells expressing recombinant TCR with target cells expressing HPV 16 E7 labeled with NucLight Red (NLR).

FIG. 4B shows cytolytic activity and interleukin production by TCR-expressing CD4+ T cells, as measured by culturing effector cells expressing recombinant TCR with target cells expressing HPV 16 E7 labeled with NucLight Red (NLR).

Figures 5A-5B show the in vivo anti-tumor activity of CD4+ and CD8+ human T cells expressing recombinant TCR 57, as assessed by administration of the engineered cells in a mouse tumor model. Figure 5A shows the mean tumor volume and Figure 5B shows the tumor volume of individual mice/groups.

Figures 6A-6B depict cell surface expression of Vβ, CD8, and MHC/E7(11-19) peptide tetramer binding in primary human CD4+ and CD8+ T cells expressing recombinant TCR 57 or reference TCR (E7 reference), as assessed by flow cytometry and compared to mock controls ( Figure 6A ). Figure 6B depicts the percentage of Vβ-positive CD8+ and CD4+ T cells in each group.

FIG. 7A depicts the cytolytic activity of CD8+ T cells expressing recombinant TCR57, reference TCR (E7 reference), and mock controls, indicated by the loss of red fluorescently labeled target cells using the IncuCyte® Live Cell Analysis System (Essen Bioscience). Figures 7B to 7D depict the cytolytic activity of CD4+ ( Figures 7B and 7D ) and CD8+ ( Figures 7B and 7C ) T cells expressing recombinant TCR 57 or reference TCR (E7 reference), as represented by the area under the curve (AUC) of the percentage of target cell killing after co-culture with various NucLight Red (NLR)-labeled target cells (SCC152, SCC090, CaSKi) expressing HPV 16 E7. Figure 7E shows interferon-γ (IFN-γ) production by CD8+ and CD4+ T cells after co-culture with target cells SCC152, SCC090, CaSKi and K562-E7 (K562 cells expressing E7) expressing HPV E7. K562-E6 (expressing nonspecific antigens) or SiHa squamous cell carcinoma cells expressing HPV16 E7 but not HLA-A02:01 were evaluated as controls.

FIG8 shows the in vivo anti-tumor activity of CD4+ and CD8+ human T cells expressing recombinant TCR 57, as shown after administration of the engineered cells in a mouse tumor model and expressed as mean tumor volume over time.

FIG. 9 shows the results of peptide titration used to determine the sensitivity of TCR 57 to non-target peptides Peptide A and Peptide B.

Cross-references to related applications

This application claims priority to U.S. Provisional Application No. 62/653,516 filed on April 5, 2018, entitled “T CELL RECEPTORS AND ENGINEERED CELLS EXPRESSING SAME” and U.S. Provisional Application No. 62/739,145 filed on September 28, 2018, entitled “T CELL RECEPTORS AND ENGINEERED CELLS EXPRESSING SAME”, the contents of which are incorporated by reference in their entirety.

The sequence listing is incorporated by reference

This application is filed with a sequence listing in electronic format. The sequence listing is provided as a 664 kilobyte file named 735042015142SeqList.txt created on March 31, 2019. The information in the electronic sequence listing is incorporated by reference in its entirety.

Provided are binding molecules, such as T cell receptors (TCRs), for example, binding molecules that bind or recognize peptide antigenic determinants associated with cancer antigens in the context of MHC molecules, such as peptide antigenic determinants expressed on the surface of cancer cells and/or cells infected with human papillomavirus (HPV) or containing HPV DNA sequences. Provided are methods suitable for treating such diseases and conditions and/or targeting such cell types, such as cancer cells or cells infected with HPV. In some embodiments, provided are binding molecules, such as TCRs, that bind or recognize peptide antigenic determinants of HPV 16 E6 or E7 in the context of MHC molecules. Such binding molecules include T cell receptors (TCRs) and their antigen-binding fragments and antibodies and their antigen-binding fragments, which exhibit specific binding or recognition to peptide antigenic determinants of HPV 16 E6 or E7.

Also provided are nucleic acid molecules encoding binding molecules, engineered cells containing binding molecules, compositions containing binding molecules or cells, and therapeutic methods including administration of such binding molecules, engineered cells or compositions, and uses of such binding molecules, cells or compositions. In some embodiments, engineered cells expressing the provided binding molecules (e.g., TCRs or antigen-binding fragments thereof) exhibit cytotoxic activity against target cells expressing peptide antigenic determinants, such as cancer cells or cells infected with HPV.

Donor cell therapy (including donor cell therapy involving the administration of cells expressing recombinant receptors or binding molecules specific for the disease or condition of interest, such as recombinant T cell receptors (TCRs) and/or other recombinant antigen receptors, such as chimeric antigen receptors, and other donor immune cells and donor T cell therapy) can be effective in treating cancer and other diseases and conditions. In certain circumstances, the methods available for donor cell therapy may not always be completely satisfactory. In some cases, optimal efficacy may depend on the ability of the administered cells to express the recombinant receptor; and the ability of the recombinant receptor to recognize and bind to a target within the individual, tumor, and its environment, such as a target antigen, such as a peptide epitope of HPV 16 E6 or E7; and uniform, homogenous, and/or consistent expression of the receptor in cells, such as in an immune cell population and/or cells in a therapeutic cell composition.

In some cases, the optimal response to a therapy may depend on the ability of an engineered recombinant receptor (e.g., TCR) to consistently and reliably express and/or bind to a target antigen on the cell surface. For example, in some cases, the properties of certain recombinant receptors (e.g., TCR) may affect the expression and/or activity of the recombinant receptor when expressed in cells (e.g., human T cells) used in cell therapy. In some cases, the expression amount of a particular recombinant receptor (e.g., TCR) may be low, and the activity of an engineered cell (e.g., human T cell) expressing such a recombinant receptor may be limited due to poor expression or poor signaling activity. In some cases, the consistency and/or efficiency of expression of recombinant receptors and receptor activity in certain cells or certain cell populations is limiting for use in therapeutic approaches. In some cases, large numbers of engineered T cells (high antigen:target (E:T) ratios) are required to exhibit functional activity.

In some aspects, the development of humanized and/or fully human recombinant TCRs presents technical challenges. For example, in some aspects, humanized and/or fully human recombinant TCR receptors compete with endogenous TCR complexes and/or can form mispairing with endogenous TCR alpha and/or TCR beta chains, which in some aspects can reduce recombinant TCR signaling, activity and/or expression, and ultimately lead to reduced activity of the engineered cells. For example, in some cases, for signaling molecules and/or domains, such as the availability of invariant CD3 signaling molecules (e.g., co-expression of CD3 delta, epsilon, gamma, and zeta chains), which are involved in allowing complex expression on the cell surface, suboptimal expression of the engineered or recombinant TCR may occur due to competition with endogenous TCRs and/or with TCRs with mispaired chains. In some aspects, the available CD3 zeta molecules can limit the expression and function of the TCR in the cell.

In some cases, certain available recombinant receptors, such as TCRs, exhibit poor expression or activity, due in part to mispairing and/or competition with endogenous TCR chains and/or other factors. In some cases, negative effects of endogenous human TCR chains on certain TCRs have been observed, including with respect to the expression, activity and/or function of such recombinant TCRs when expressed in human T cells.

One approach to addressing these challenges is to design recombinant TCRs containing mouse constant domains to prevent mispairing with endogenous human TCR alpha or TCR beta chains. For example, to avoid such problems, certain reference TCRs, such as those described in International PCT Publication No. WO 2015/184228, have been engineered by using a combination of mouse constant regions and reference TCR variable regions. However, the use of recombinant TCRs containing mouse sequences may present an immune response risk in some embodiments. Other approaches to addressing these challenges include introducing gene interruptions at endogenous genes encoding one or more TCR chains, such as by gene editing.

In some aspects, the recombinant TCRs provided are TCRs that allow for similar or improved performance and/or activity compared to other available recombinant TCRs without the need for methods such as engineering with mouse constant domains and/or introducing gene interruptions at endogenous genes encoding one or more TCR chains. In some aspects, the embodiments provided are based on the observation that certain fully human recombinant TCRs specific for HPV E7 have improved performance and activity without the use of mouse constant domains or without the introduction of gene interruptions at endogenous TCR encoding genes. In some aspects, the recombinant TCRs provided, including exemplary TCRs, are fully human TCRs containing human constant domains. In some aspects, provided embodiments provide the advantages of recombinant TCRs that exhibit high and consistent expression and improved functional activity in human T cells, even in the presence of endogenous TCRs.

In some aspects, the recombinant TCR provided comprises a TCR that is at least partially CD8-independent. In some cases, TCR recognition of a peptide in the context of an MHC molecule and subsequent T cell activation is promoted in the presence of a CD8 co-receptor. For example, engagement of a CD8 co-receptor can promote low to moderate TCR affinity interactions and/or T cell activation (see, e.g., Kerry et al. J. Immunology (2003) 171(9): 4493-4503; and Robbins et al. J Immunology (2008) 180(9): 6116-6131). The binding molecules provided are molecules, such as TCRs, that exhibit CD8-independent binding to HPV E6 or E7 peptide epitopes. Such binding molecules, such as TCRs, can provide the following advantages: Functional avidity or affinity is higher than that of TCRs or antigen-binding fragments thereof that require the presence of CD8 co-expression. In some embodiments, the provided CD8-independent binding molecules, such as TCRs, can be expressed or engineered in cells that do not express CD8 (e.g., T cells), such as CD4+ cells. In some embodiments, CD4+ cells engineered with the provided TCRs do not require CD8 co-receptors to exhibit functional activity.

In some aspects, the embodiments provided are based on the observation that the performance and activity of exemplary fully human recombinant TCRs specific for HPV E7 are improved even at low effector:target (E:T) ratios. In some cases, the activity of engineered T cells expressing the recombinant receptor, such as cytolytic activity, is limited when fewer engineered T cells are present compared to target cells (e.g., cancer cells expressing the antigen). In some aspects, such improved activity, particularly at low E:T ratios and when using fully human sequences, is beneficial for improving the efficacy of therapies such as donor-receiver cell therapy.

In some cases, certain available methods for obtaining antigen-specific recombinant receptors (such as recombinant TCRs) can produce recombinant receptors that exhibit cross-reactivity to different non-target antigens (see, e.g., Cameron et al., (2013) Science Translational Medicine, 5(197): 197ra103). In some aspects, the embodiments provided are based on the observation that, as described herein, the TCRs provided are fully human recombinant TCRs specific for HPV E7 that do not show cross-reactivity to cells expressing other peptide antigens or alloreactivity to other human leukocyte antigen (HLA) subtypes. The provided TCRs thus exhibit improved performance and activity with minimal risk of cross-reactivity to other antigens that may be present in an individual (e.g., non-target antigens) or peptide epitopes present on non-target HLA subtypes.

In some embodiments, therapeutic methods using such TCRs, such as acceptor cell therapy using engineered human T cells expressing the provided recombinant TCRs, can ultimately produce high efficacy while minimizing the risk of immunogenicity from using mouse constant regions or the risk of cross-reactivity or off-target reactivity, requiring additional engineering steps. In some cases, provided embodiments, including recombinant receptors, polynucleotides encoding such receptors, engineered cells, and cell compositions, can provide various advantages over available therapies using recombinant receptors to improve the activity of recombinant receptors and the response to acceptor cell therapy targeting HPV-related cancers.

All publications, including patent documents, scientific papers, and databases, cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein shall control and not the definition incorporated herein by reference.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

I. T cell receptors and binding molecules

Provided herein are binding molecules, such as binding molecules that bind or recognize peptide antigenic determinants associated with cancer antigens in the context of MHC molecules, such as peptide antigenic determinants expressed on the surface of cancer cells and/or cells infected with human papillomavirus (HPV) or containing HPV DNA sequences. In some embodiments, the provided binding molecules bind or recognize peptide antigenic determinants of HPV 16 E6 or E7 in the context of MHC molecules. In some embodiments, the binding molecules bind or recognize peptide antigenic determinants of antigens expressed on the surface of cancer cells or immortalized cell lines such as the exemplary cell line SCC152. Such binding molecules include T cell receptors (TCRs) and antigen binding fragments thereof and antibodies and antigen binding fragments thereof, which exhibit antigenic specificity for binding or recognizing such peptide antigenic determinants. In some embodiments, nucleic acid molecules encoding binding molecules, engineered cells containing binding molecules, compositions and treatment methods are also provided, which methods involve administering such binding molecules, engineered cells or compositions. In some aspects, engineered cells expressing the provided binding molecules, such as TCRs or antigen binding fragments, exhibit cytotoxic activity against target cells expressing peptide antigenic determinants, such as cancer cells or cells infected with HPV or containing HPV DNA sequences, such as the exemplary cell line SCC152.

HPV is the causative organism in most cases of cervical cancer and has been associated with anal, vaginal, vulvar, penile, and oropharyngeal cancers, as well as other cancers. Typically, the HPV genome contains an early region containing six open reading frames (E1, E2, E4, E5, E6, and E7) that encode proteins involved in cell transformation and replication, and a late region containing two open reading frames (L1 and L2) that encode proteins of the viral capsid. In general, E6 and E7 are oncogenes that can affect cell cycle regulation and contribute to the formation of cancer. For example, the E6 gene product can cause p53 degradation, and the E7 gene product can cause retinoblastoma (Rb) inactivation.

In some aspects, the binding molecules provided include TCR or its antigen-binding fragment or antibody, such as its antibody fragment, and protein, such as chimeric molecules containing one or more of the above, such as chimeric receptors, such as TCR-like CAR, and/or engineered cells expressing TCR or CAR, and peptide antigenic determinants derived from HPV16 E6 protein, HPV16 E7 protein and/or peptide antigenic determinants expressed on HPV-infected cells. In some embodiments, the binding molecule is an anti-HPV-16 binding molecule, such as an anti-HPV16 E6 binding molecule or an anti-HPV16 E7 binding molecule.

In some aspects, the binding molecule recognizes or binds to an HPV 16 E6 or E7 antigenic determinant in the context of an MHC molecule, such as an MHC class I molecule. In some aspects, the MHC class I molecule is a human leukocyte antigen (HLA)-A2 molecule, including any one or more subtypes thereof, such as HLA-A*0201, *0202, *0203, *0206, or *0207. In some cases, there may be differences in the frequency of subtypes between different populations. For example, in some embodiments, more than 95% of HLA-A2 positive Caucasian populations are HLA-A*0201, whereas in Chinese populations, the frequencies are reported to be approximately 23% HLA-A*0201, 45% HLA-A*0207, 8% HLA-A*0206, and 23% HLA-A*0203. In some embodiments, the MHC molecule is HLA-A*0201.

In some embodiments, the TCR or antigen-binding fragment thereof recognizes or binds to an antigenic determinant or region of HPV16 E6 or HPV 16 E7, such as a peptide antigenic determinant containing the amino acid sequence shown in any one of SEQ ID NOs: 267-274, and as shown in Table 1 below.

In some embodiments, the binding molecule, such as a TCR or an antigen-binding fragment thereof or an antibody or an antigen-binding fragment thereof, is isolated or purified or recombinant. In specific embodiments, any of the provided binding molecules, such as a TCR or an antigen-binding fragment thereof or an antibody or an antigen-binding fragment thereof, is recombinant. In some aspects, the binding molecule, such as a TCR or an antigen-binding fragment thereof or an antibody or an antigen-binding fragment thereof, is human. In some embodiments, the binding molecule is monoclonal. In some aspects, the binding molecule is single-chain. In other embodiments, the binding molecule contains two chains. In some embodiments, the binding molecule, such as a TCR or an antigen-binding fragment thereof or an antibody or an antigen-binding fragment thereof, is expressed on the surface of a cell.

In some embodiments, the binding molecules provided have one or more specified functional characteristics, such as binding properties, including binding to a specific antigenic determinant, and/or a specific binding affinity as described.

A. T cell receptor (TCR)

In some embodiments, the binding molecule provided is a T cell receptor (TCR) or an antigen binding fragment thereof. In some embodiments, a "T cell receptor" or "TCR" is a molecule containing a variable α chain and a β chain (also referred to as TCRα and TCRβ, respectively) or a variable γ chain and a δ chain (also referred to as TCRγ and TCRδ, respectively) or an antigen binding portion thereof, and capable of specifically binding to a peptide bound to an MHC molecule. In some embodiments, the TCR is in the αβ form. Generally, TCRs in the αβ and γδ forms are generally similar in structure, but the T cells expressing them may have different anatomical locations or functions. TCRs may be found on the surface of cells or in soluble form. Generally speaking, TCRs are found on the surface of T cells (or T lymphocytes) and are generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

Unless otherwise indicated, the term "TCR" should be understood to cover the full TCR as well as its antigen-binding portion or antigen-binding fragment. In some embodiments, the TCR is a complete or full-length TCR, such as a TCR containing an alpha chain and a beta chain. In some embodiments, the TCR is an antigen-binding portion that is smaller than the full-length TCR but binds to a specific peptide bound in an MHC molecule, such as an MHC-peptide complex. In some cases, the antigen-binding portion or fragment of a TCR may contain only a portion of the domain of the full-length or complete TCR, but is still able to bind to a peptide antigenic determinant, such as an MHC-peptide complex, bound by the full TCR. In some cases, the antigen binding portion contains the variable domains of a TCR, such as the variable α ( _Vα ) chain and the variable β ( _Vβ ) chain of a TCR, or an antigen binding fragment thereof sufficient to form a binding site for binding to a specific MHC-peptide complex.

In some embodiments, the variable domain of a TCR contains complementary determining regions (CDRs), which are typically the major contributors to antigen recognition and binding capacity and specificity of peptides, MHC, and/or MHC-peptide complexes. In some embodiments, the CDRs of a TCR or a combination thereof form all or substantially all of the antigen binding sites of a given TCR molecule. Each CDR within the variable region of a TCR chain is typically separated by a framework region (FR), which typically exhibits lower variability among TCR molecules than the CDRs (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. USA 87 : 9138, 1990; Chothia et al. EMBO J. 7 : 3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27 : 55, 2003). In some embodiments, CDR3 is the primary CDR responsible for antigen binding or specificity or is the most important of the three CDRs on a given TCR variable region for antigen recognition and/or for interaction with the processed peptide portion of a peptide-MHC complex. In some cases, CDR1 of the alpha chain can interact with the N-terminal portion of certain antigenic peptides. In some cases, CDR1 of the beta chain can interact with the C-terminal portion of a peptide. In some cases, CDR2 contributes most to or is the primary CDR responsible for interaction with or recognition of the MHC portion of an MHC-peptide complex. In some embodiments, the variable region of the β chain may contain another hypervariable region (CDR4 or HVR4), which is usually involved in superantigen binding but not in antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8: 411-426).

In some embodiments, the alpha chain and/or beta chain of the TCR may also contain a constant domain, a transmembrane domain, and/or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease , 3rd ed., Current Biology Publications, p. 4:33, 1997). In some aspects, each chain of the TCR (e.g., alpha or beta) may have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end. In some embodiments, the TCR, for example, via the cytoplasmic tail, binds to an invariant protein of the CD3 complex that is involved in mediating signal transduction. In some cases, the structure allows the TCR to bind to other molecules, such as CD3 and its subunits. For example, a TCR containing a homeostatic domain and a transmembrane region can anchor the protein in the cell membrane and associate with an invariant subunit of the CD3 signaling machinery or complex. The intracellular tail of the CD3 signaling subunit (e.g., CD3γ, CD3δ, CD3ε, and CD3ζ chains) contains one or more immunoreceptor tyrosine-based activation motifs or ITAMs and is generally involved in the signaling ability of the TCR complex.

It is within the capabilities of those skilled in the art to determine or identify various domains or regions of a TCR. In some cases, the exact locus of a domain or region may vary depending on the specific structure or homology modeling or other features used to describe a specific domain. It should be understood that reference to amino acids, including specific sequences shown in SEQ ID NOs used to describe the domain organization of a TCR, is for the purpose of illustration and is not intended to limit the scope of the embodiments provided. In some cases, a specific domain (e.g., a variable domain or a constant domain) may be longer or shorter by several amino acids (e.g., one, two, three, or four). In some aspects, the residues of the TCR are known or can be identified according to the International Immunogenetics Information System (IMGT) numbering system (see, e.g., www.imgt.org; see also Lefranc et al. (2003) Developmental and Comparative Immunology, 27(1); 55-77; and The T Cell Factsbook 2nd ed., Lefranc and LeFranc Academic Press 2001). Using this system, the CDR1 sequence within the TCR Vα chain and/or Vβ chain corresponds to the amino acids present between and including residue numbers 27-38, the CDR2 sequence within the TCR Vα chain and/or Vβ chain corresponds to the amino acids present between and including residue numbers 56-65, and the CDR3 sequence within the TCR Vα chain and/or Vβ chain corresponds to the amino acids present between and including residue numbers 105-117.

In some embodiments, the α chain and the β chain of the TCR each further contain a constant domain. In some embodiments, the α chain constant domain (Cα) and the β chain constant domain (Cβ) are individually mammalian, such as human or mouse constant domains. In some embodiments, the constant domain is adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains and two membrane-distal variable domains, each of which contains CDRs.

In some aspects, provided herein are TCRs containing human constant regions, such as an alpha chain containing a human Cα region and a beta chain containing a human Cβ. In some embodiments, the provided TCRs are fully human. The provided TCRs are TCRs containing human constant regions, such as fully human TCRs, whose expression and/or activity, such as when expressed in human cells, such as human T cells, such as primary human T cells, is not affected or substantially not affected by the presence of endogenous human TCRs. In particular, it is observed herein that certain TCRs, such as the exemplary TCR designated as TCR 57; or a TCR containing Vα as shown in SEQ ID NO: 47 and Vβ as shown in SEQ ID NO: 60; or a TCR containing the CDRs as shown in SEQ ID NO: 48, 49, 50, 61, 62, and 63, when formatted with a human constant region, exhibit substantial activity in primary human T cells containing endogenous TCRs. In some embodiments, such TCRs containing human constant regions are not knocked out by endogenous human TCRs, such as for components of the CD3 complex.

In some embodiments, such TCRs containing a human constant region, when expressed by a human cell, such as a human T cell, that contains or expresses an endogenous human TCR, are expressed on the cell surface in an amount that is similar or improved, exhibit similar or greater functional activity (e.g., cytolytic activity), and/or exhibit similar or greater anti-tumor activity, compared to the amount of expression, functional activity, and/or anti-tumor activity of the same TCR in a similar human cell in which expression of the endogenous TCR has been reduced or eliminated. In some examples, a TCR containing a human constant region provided herein, when expressed in a human T cell, is expressed on the cell surface at an amount that is at least or about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% or 120% of the amount expressed when the same TCR is expressed in a similar human T cell in which expression of the endogenous TCR has been reduced or eliminated. In some examples, TCRs containing a human constant region provided herein, when expressed in human T cells, exhibit antigen-dependent functional activity, such as cytolytic activity, that is at least or about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% or 120% of the activity of the same TCR expressed in similar human cells but in which expression of the endogenous TCR has been reduced or eliminated. In some examples, a TCR containing a human constant region provided herein, when expressed in human T cells, exhibits anti-tumor activity, such as when administered to an individual in vivo, the anti-tumor activity is at least or about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% or 120% of the anti-tumor activity of the same TCR when expressed in similar human cells, but in which expression of the endogenous TCR has been reduced or eliminated. In some of any of the above embodiments, cells in which expression of an endogenous TCR has been reduced or eliminated can include cells in which genes and/or gene products encoding the TCR, such as TRAC and/or TRBC , are reduced, deleted, eliminated, knocked out or interrupted, such as by any of a variety of gene editing methods.

In some embodiments, such TCRs containing human constant regions, when expressed by human cells, such as human T cells, containing or expressing endogenous human TCRs, are expressed on the cell surface at a similar or improved amount, exhibit similar or greater functional activity (e.g., cytolytic activity), and/or exhibit similar or greater anti-tumor activity, as compared to the amount, functional activity, and/or anti-tumor activity of a similar TCR containing the same Vβ and Vα regions but formatted with mouse constant regions when expressed in human cells. In some examples, a TCR containing a human constant region provided herein, when expressed in human T cells, is expressed on the cell surface at an amount that is at least or about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, or 120% of the amount expressed when expressed in human T cells by a similar TCR containing the same Vβ and Vα regions but formatted with mouse constant regions. In some examples, a TCR containing a human constant region provided herein, when expressed in human T cells, exhibits an antigen-dependent functional activity, such as cytolytic activity, that is at least or about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% or 120% of the activity of a similar TCR containing the same Vβ and Vα regions but formatted with mouse constant regions when expressed in human T cells. In some examples, a TCR containing a human constant region provided herein, when expressed in human T cells, exhibits anti-tumor activity, such as when administered to an individual in vivo, the anti-tumor activity is at least or about 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115% or 120% of the anti-tumor activity of a similar TCR containing the same Vβ and Vα regions but formatted with mouse constant regions when expressed in human T cells.

In some embodiments, each of the Cα and Cβ domains is human. In some embodiments, Cα is encoded by the TRAC gene (IMGT nomenclature) or is a variant thereof. In some embodiments, the variant of Cα contains at least one non-natural cysteine substitution, such as any substitution described herein. In some embodiments, Ca or a variant thereof has or comprises the amino acid sequence shown in SEQ ID NO: 14, 333, 334, 335, 336, 337, 338, 341, 344, 345, 346, 347 or 348, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 14, 333, 334, 335, 336, 337, 338, 341, 344, 345, 346, 347 or 348. In some embodiments, Ca has or comprises the amino acid sequence shown in SEQ ID NO: 14. In some embodiments, Cβ is encoded by TRBC1 or TRBC2 gene (IMGT nomenclature) or is a variant thereof. In some embodiments, the variant of Cβ contains at least one non-natural cysteine substitution, such as any substitution described herein. In some embodiments, Cβ or a variant thereof has or comprises the amino acid sequence set forth in SEQ ID NO: 29, 339, 340, 342, 343, 349, 350, 351 or 352, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 29, 339, 340, 342, 343, 349, 350, 351 or 352. In some embodiments, Cβ has or includes the amino acid sequence shown in SEQ ID NO: 29. In some embodiments, Cβ has or includes the amino acid sequence shown in SEQ ID NO: 350.

In some embodiments, Cα is or comprises the amino acid sequence shown in SEQ ID NO: 14, 333, 334, 335, 336, 337, 338, 341, 344, 345, 346, 347 or 348, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 14, 333, 334, 335, 336, 337, 338, 341, 344, 345, 346, 347 or 348, and/or Cβ is or comprises the amino acid sequence shown in SEQ ID NO: 29, 339, 340, 342, 343, 350, 351 or 352, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: NO: 29, 339, 340, 342, 343, 350, 351 or 352 exhibits an amino acid sequence of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity.

In some embodiments, any of the provided TCRs or antigen-binding fragments thereof may be human/mouse chimeric TCRs. In some cases, the TCR or antigen-binding fragment thereof comprises an alpha chain and/or a beta chain comprising a mouse constant region. In some embodiments, Cα is a mouse constant region, which is or comprises the amino acid sequence shown in SEQ ID NO: 15 or 275-282 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with SEQ ID NO: 15 or 275-282. In some embodiments, Cα is or comprises the amino acid sequence shown in SEQ ID NO: 15 or 275-282. In some embodiments, Cβ is a mouse constant region, which is or comprises the amino acid sequence shown in SEQ ID NO: 30 or 283-285 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with SEQ ID NO: 30 or 283-285. In some embodiments, Cβ is or comprises the amino acid sequence shown in SEQ ID NO: 30 or 283-285.

In some embodiments, Ca is or comprises the amino acid sequence set forth in SEQ ID NO: 275, 276 or 279, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 275, 276 or 279, and/or Cβ is or comprises the amino acid sequence set forth in SEQ ID NO: 283 or 284, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 283 or 284. In some embodiments, Cα and/or Cβ is or comprises any Cα and/or Cβ described in WO 2015/184228, WO 2015/009604 and WO 2015/009606.

In some embodiments, a TCR or antigen-binding fragment thereof comprises a variant of the alpha chain and/or the beta chain. In some embodiments, the variant comprises an amino acid sequence of any of the TCRs described herein, which contains one, two, three, or four or more amino acid substitutions in the constant region of the alpha chain or the beta chain. In some embodiments, a TCR (or a functional portion thereof) comprising a substituted amino acid sequence advantageously provides one or more of the following: reduced mispairing with endogenous TCR chains, increased expression in host cells, increased recognition of HPV 16 targets, and increased anti-tumor activity compared to a parent TCR comprising an unsubstituted amino acid sequence.

In some embodiments, the constant region contains a substituted amino acid sequence of a mouse constant region of TCR α chain and β chain, which corresponds to all or part of an unsubstituted mouse constant region α amino acid sequence (e.g., SEQ ID NO: 275, 276, or 279) and β amino acid sequence (e.g., 282, 283, 284), respectively. In some embodiments, the constant region is a substituted amino acid sequence of a mouse constant region TCR α chain and β chain as shown in SEQ ID NO: 280 and 285, respectively, wherein SEQ ID NO: 280 has one, two, three, or four amino acid substitutions when compared to SEQ ID NO: 279, and SEQ ID NO: 285 has one amino acid substitution when compared to SEQ ID NO: 284. In some embodiments, the variant of the TCR comprises the following amino acid sequence: (a) SEQ ID NO: 280 (constant region of α chain), wherein (i) X at position 48 is Thr or Cys; (ii) X at position 112 is Ser, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; (iii) X at position 114 is Met, Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; and (iv) X at position 115 is Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; and (b) SEQ ID NO: 285 (constant region of β chain), wherein X at position 56 is Ser or Cys. In some embodiments, Cα is or comprises the amino acid sequence set forth in SEQ ID NO: 280 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 280, and/or Cβ is or comprises the amino acid sequence set forth in SEQ ID NO: 285 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 285.

In some embodiments, the TCR may be a heterodimer of two chains, α and β, connected by one or more disulfide bonds. In some embodiments, the constant domain of the TCR may contain a short linker sequence in which cysteine residues form a disulfide bond, thereby connecting the two chains of the TCR. In some embodiments, the TCR may have additional cysteine residues in each of the α chain and the β chain, so that the TCR contains two disulfide bonds in the constant domain. In some embodiments, the constant domain and the variable domain each contain a disulfide bond formed by a cysteine residue.

In some embodiments, the TCR may contain one or more introduced disulfide bonds. In some embodiments, there are no native disulfide bonds. In some embodiments, one or more of the native cysteines (e.g., in the constant domains of the α and β chains) that form the native interchain disulfide bonds are substituted with another residue, such as serine or alanine. In some embodiments, the introduced disulfide bonds may be formed by mutating non-cysteine residues on the α and β chains, such as in the constant domains of the α and β chains, to cysteine. The opposing cysteines in the TCR alpha and beta chains provide disulfide bonds that link the constant regions of the TCR alpha and beta chains of the substituted TCR to each other and are not present in a TCR comprising an unsubstituted constant region, in which natural disulfide bonds are present, such as an unsubstituted natural human constant region or an unsubstituted natural mouse constant region. In some embodiments, the presence of non-natural cysteine residues in the recombinant TCR (e.g., resulting in one or more non-natural disulfide bonds) facilitates the production of the desired recombinant TCR in a cell into which it is introduced rather than expressing a mismatched TCR pair containing a natural TCR chain.

Exemplary non-natural disulfide bonds of TCRs are described in International Publication Nos. WO2006/000830 and WO2006/037960. In some embodiments, cysteine can be introduced or substituted at the following positions: at the residues corresponding to Thr48 of the Cα chain and Ser57 of the Cβ chain; at the residue Thr45 of the Cα chain and the residue Ser77 of the Cβ chain; at the residue Tyr10 of the Cα chain and the residue Ser17 of the Cβ chain; at the residue Thr45 of the Cα chain and the residue Asp59 of the Cβ chain; and/or at the residue Ser15 of the Cα chain and the residue Glu15 of the Cβ chain, with reference to the numbering of the Cα shown in any one of SEQ ID NOs: 333-335 or 337 or the Cβ shown in SEQ ID NOs: 339 or 340.

In some embodiments, any of the provided cysteine mutations can be made at corresponding positions in another sequence, such as in the human or mouse Cα and Cβ sequences described above. The term "corresponding" with respect to protein positions is as follows: An amino acid position "corresponding to" an amino acid position in a disclosed sequence, such as shown in the sequence listing, refers to an amino acid position identified when aligned to the disclosed sequence based on structural sequence alignment or using a standard alignment algorithm, such as the GAP algorithm. For example, corresponding residues can be determined by aligning a reference sequence with the Cα sequence shown in any one of SEQ ID NOs: 333, 334, 335, 336, or 337 or the Cβ sequence shown in SEQ ID NOs: 339 or 340 by structural alignment methods as described herein. By aligning the sequences, one skilled in the art can, for example, use conserved and identical amino acid residues as a guide to identify corresponding residues. For example, Thr48 in the Cα chain aligns or corresponds to Thr49 in the sequence shown in SEQ ID NO: 338 or 341, and Ser57 in the Cβ chain aligns or corresponds to Ser58 in the sequence shown in SEQ ID NO: 342 or 343.

In some embodiments, the constant region is substituted with cysteine at positions corresponding to positions Thr48 and Ser57 as described above compared to the native human constant region. In some embodiments, the variant of the TCR is substituted with cysteine, wherein one or both of the native Thr48 of SEQ ID NO: 334 and the native Ser57 of SEQ ID NO: 339 are substituted with Cys. In some embodiments, Ca is or comprises the amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 14, and/or Cβ is or comprises the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 29. In some embodiments, the TCR comprises the Ca chain and the Cβ chain as set forth in SEQ ID NOs: 14 and 29, respectively. In some embodiments, Cα is or comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with SEQ ID NO: 14, and/or Cβ is or comprises the amino acid sequence shown in SEQ ID NO: 350 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with SEQ ID NO: 350. In some embodiments, the TCR comprises the Cα chain and the Cβ chain as shown in SEQ ID NO: 14 and 350, respectively.

In some embodiments, the constant region is substituted with cysteine at positions corresponding to positions Thr48 and Ser57 as described above compared to the native mouse constant region. In some embodiments, the variant of the TCR is a cysteine-substituted chimeric TCR, wherein one or both of the native Thr48 of SEQ ID NO: 279 and the native Ser57 of SEQ ID NO: 284 are substituted with Cys. In some embodiments, Ca is or comprises the amino acid sequence set forth in SEQ ID NO: 15 or 281, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 15 or 281, and/or Cβ is or comprises the amino acid sequence set forth in SEQ ID NO: 30, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 30. In some embodiments, the TCR comprises the Ca chain and the Cβ chain as set forth in SEQ ID NO: 15 and 30, respectively. In some embodiments, the TCR comprises a Cα chain and a Cβ chain as shown in SEQ ID NOs: 281 and 30, respectively.

In some embodiments, the variant comprises a hydrophobic amino acid substitution for one, two or three amino acids in the transmembrane (TM) domain of one or both of the constant regions of the α chain and the β chain, resulting in a TCR substituted with a hydrophobic amino acid. The hydrophobic amino acid substitution in the TM domain of the TCR can increase the hydrophobicity of the TM domain of the TCR compared to a TCR without a hydrophobic amino acid substitution in the TM domain. In some embodiments, the variant of the TCR comprises one, two or three of the native Ser 112, Met 114 and Gly 115 of SEQ ID NO: 279, which can be independently substituted with Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp; for example, substituted with Leu, Ile or Val. In some embodiments, Cα is or comprises the amino acid sequence set forth in SEQ ID NO: 282 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with SEQ ID NO: 282, and/or Cβ is or comprises the amino acid sequence set forth in SEQ ID NO: 284 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with SEQ ID NO: 284.

In some embodiments, the variants include a combination of cysteine substitutions in the constant region of one or both of the α chain and the β chain and substitutions of one, two or three amino acids in the transmembrane (TM) domain of the constant region of one or both of the α chain and the β chain with hydrophobic amino acids. In some embodiments, the variants have the native Thr48 of SEQ ID NO: 279 substituted with Cys; one, two or three of the native Ser 112, Met 114 and Gly 115 of SEQ ID NO: 279 are independently substituted with Gly, Ala, Val, Leu, Ile, Pro, Phe, Met or Trp, such as Leu, Ile or Val; and the native Ser57 of SEQ ID NO: 284 is substituted with Cys. In some embodiments, Cα is or comprises the amino acid sequence set forth in SEQ ID NO: 277 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 277, and/or Cβ is or comprises the amino acid sequence set forth in SEQ ID NO: 30 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 30.

Exemplary sequences of TCRs provided (e.g., CDR, Vα and/or Vβ and constant region sequences) are described herein, e.g., in Section I.A.1 below.

In some embodiments, the TCR is a full-length TCR. In some embodiments, the TCR is an antigen-binding portion. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR (sc-TCR). The TCR may be cell-bound or in soluble form. In some embodiments, the TCR is expressed on the surface of a cell in a cell-bound form.

In some embodiments, the dTCR contains a first polypeptide in which a sequence corresponding to a provided TCR α chain variable region sequence is fused to the N-terminus of a sequence corresponding to an extracellular sequence of a constant region of a TCR α chain; and a second polypeptide in which a sequence corresponding to a provided TCR β chain variable region sequence is fused to the N-terminus of a sequence corresponding to an extracellular sequence of a constant region of a TCR β chain, and the first polypeptide and the second polypeptide are linked by a disulfide bond. In some embodiments, the bond can correspond to a natural inter-chain disulfide bond present in a natural dimeric αβ TCR. In some embodiments, there is no inter-chain disulfide bond in a natural TCR. For example, in some embodiments, one or more cysteines can be incorporated into the extracellular sequence of the constant region of the dTCR polypeptide pair. In some cases, both natural and non-natural disulfide bonds are desired. In some embodiments, the TCR contains a transmembrane sequence to anchor to the membrane.

In some embodiments, the dTCR contains a provided TCR α chain, which contains a variable α domain, a constant α domain, and a first dimerization motif connected to the C-terminus of the constant α domain; and a provided TCR β chain, which contains a variable β domain, a constant β domain, and a first dimerization motif connected to the C-terminus of the constant β domain, wherein the first and second dimerization motifs are prone to interact with each other, forming a covalent bond between the amino acid in the first dimerization motif and the amino acid in the second dimerization motif, connecting the TCR α chain and the TCR β chain together.

In some embodiments, the TCR is a scTCR, which is a single amino acid chain containing an alpha chain and a beta chain capable of binding to an MHC-peptide complex. Generally, scTCRs can be produced using methods known to those skilled in the art, see, for example, International Publication Nos. WO 96/13593, WO 96/18105, WO99/18129, WO 04/033685, WO2006/037960, WO2011/044186; U.S. Patent No. 7,569,664; and Schlueter, C.J. et al. J. Mol. Biol. 256, 859 (1996).

In some embodiments, the scTCR contains a first segment consisting of an amino acid sequence corresponding to the sequence of the variable region of the provided TCR α chain; a second segment consisting of an amino acid sequence corresponding to the sequence of the variable region of the provided TCR β chain, which is fused to the N-terminus of an amino acid sequence corresponding to the extracellular sequence of the constant domain of the TCR β chain; and a linker sequence that connects the C-terminus of the first segment to the N-terminus of the second segment.

In some embodiments, the scTCR contains a first segment consisting of an amino acid sequence corresponding to a provided TCR β chain variable region; a second segment consisting of an amino acid sequence corresponding to a provided TCR α chain variable region sequence fused to the N-terminus of an amino acid sequence corresponding to an extracellular sequence of a TCR α chain constant domain; and a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

In some embodiments, the scTCR contains a first segment consisting of a provided α chain variable region sequence fused to the N-terminus of the α chain extracellular homeostatic domain sequence; and a second segment consisting of a provided β chain variable region sequence fused to the N-terminus of the β chain extracellular homeostatic and transmembrane sequence; and optionally, a linker sequence that connects the C-terminus of the first segment to the N-terminus of the second segment.

In some embodiments, the scTCR contains a first segment consisting of a provided TCR β chain variable region sequence fused to the N-terminus of the β chain extracellular homeostatic domain sequence; and a second segment consisting of a provided α chain variable region sequence fused to the N-terminus of the α chain extracellular homeostatic and transmembrane sequence; and optionally, a linker sequence that connects the C-terminus of the first segment to the N-terminus of the second segment.

In some embodiments, in order for the scTCR to bind to an MHC-peptide complex, the alpha and beta chains must be paired so that their variable region sequences are oriented for such binding. Various methods for promoting the pairing of alpha and beta in a scTCR are well known in the art. In some embodiments, a linker sequence is included that connects the alpha and beta chains to form a single polypeptide chain. In some embodiments, the linker should be of sufficient length to cover the distance between the C-terminus of the alpha chain and the N-terminus of the beta chain or vice versa, while also ensuring that the linker length is not so long that it blocks or reduces the binding of the scTCR to the target peptide-MHC complex.

In some embodiments, the linker connecting the first and second TCR segments of the scTCR can be any linker that can form a single polypeptide chain while retaining the TCR binding specificity. In some embodiments, the linker sequence can, for example, have the formula -P-AA-P-, where P is proline and AA represents an amino acid sequence, where the amino acids are glycine and serine. In some embodiments, the first and second segments are paired so that their variable region sequences are oriented for such binding. Therefore, in some cases, the linker has a sufficient length to cover the distance between the C-terminus of the first segment and the N-terminus of the second segment or vice versa, but cannot be too long to block or reduce the binding of the scTCR to the target ligand. In some embodiments, the linker can contain from or about 10 to 45 amino acids, such as 10 to 30 amino acids or 26 to 41 amino acid residues, for example 29, 30, 31 or 32 amino acids. In some embodiments, the linker has the formula -PGGG-(SGGGG) _n -P-, wherein n is 5 or 6 and P is proline, G is glycine and S is serine (SEQ ID NO: 287). In some embodiments, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO: 288).

In some embodiments, the scTCR contains disulfide bonds between residues of a single amino acid chain, which in some cases can promote the stability of the pairing between the α region and the β region of the single chain molecule (see, e.g., U.S. Patent No. 7,569,664). In some embodiments, the scTCR contains covalent disulfide bonds that connect residues of the immunoglobulin region of the constant domain of the α chain of the single chain molecule to residues of the immunoglobulin region of the constant domain of the β chain. In some embodiments, the disulfide bonds correspond to the natural disulfide bonds present in the natural dTCR. In some embodiments, the disulfide bonds are not present in the natural TCR. In some embodiments, the disulfide bonds are introduced non-natural disulfide bonds, for example, by incorporating one or more cysteines into the extracellular sequence of the constant region of the first and second chain regions of the scTCR polypeptide. Exemplary cysteine mutations include any cysteine mutations as described above. In some cases, both natural and non-natural disulfide bonds may be present.

In some embodiments, the scTCR is a non-disulfide bonded truncated TCR in which a heterologous leucine zipper fused to its C-terminus promotes chain association (see, e.g., International Publication No. PCT WO99/60120). In some embodiments, the scTCR contains a TCR alpha variable domain covalently linked to a TCR beta variable domain via a peptide linker (see, e.g., International Publication No. PCT WO99/18129).

In some embodiments, any of the provided TCRs, including dTCRs or scTCRs, can be linked to a signaling domain that produces an active TCR on the surface of a T cell. In some embodiments, the TCR is expressed on the surface of a cell. In some embodiments, the TCR does contain a sequence corresponding to a transmembrane sequence. In some embodiments, the transmembrane domain is positively charged. In some embodiments, the transmembrane domain can be a Cα or Cβ transmembrane domain. In some embodiments, the transmembrane domain can be from a non-TCR source, such as a transmembrane region from CD3z, CD28, or B7.1. In some embodiments, the TCR does contain a sequence corresponding to a cytoplasmic sequence. In some embodiments, the TCR contains a CD3z signaling domain. In some embodiments, the TCR is capable of forming a TCR complex with CD3.

In some embodiments, the TCR is a soluble TCR. In some embodiments, the soluble TCR has a structure as described in WO99/60120 or WO 03/020763. In some embodiments, the TCR does not contain a sequence corresponding to a transmembrane sequence, e.g., to allow membrane anchoring to the cell in which the TCR is expressed. In some embodiments, the TCR does not contain a sequence corresponding to a cytoplasmic sequence.

1. Exemplary TCRs

Provided herein are TCRs or antigen-binding fragments thereof that recognize or bind to an antigenic determinant or region of a cancer antigen in the context of an MHC molecule, such as a peptide antigenic determinant expressed on the surface of a cancer cell and/or a cell infected with HPV or a cell containing an HPV DNA sequence. In some embodiments, the TCR or antigen-binding fragment thereof binds to or recognizes an antigen expressed on the surface of a cell line designated SCC152 (ATCC® CRL-3240 ^™ ), which is a cell line derived from squamous cell carcinoma and contains an HPV DNA sequence. In some aspects, the cytotoxic activity of T cells containing binding molecules such as TCRs is stimulated when such cells come into contact with target cells expressing antigens, such as cancer cells and/or cells expressing HPV 16, such as HPV 16 E6 or HPV 16 E7, such as SCC152 cells. In some embodiments, the TCRs provided herein or antigen-binding fragments thereof provided herein are TCRs or antigen-binding fragments thereof that bind or recognize a peptide antigenic determinant of HPV 16 (e.g., a peptide antigenic determinant of HPV 16 E6 or a peptide antigenic determinant of HPV 16 E7) in the context of MHC.

Such TCRs or antigen-binding fragments thereof are TCRs or antigen-binding fragments thereof that contain any of the variable (Vα or Vβ) region sequences of the α and/or β chains as described individually, or a sufficient antigen-binding portion of such chains. In some embodiments, the provided TCRs or antigen-binding fragments thereof (e.g., anti-HPV 16 E6 or anti-HPV 16 E7 TCRs) contain a Vα region sequence, or a sufficient antigen-binding portion thereof, containing CDR-1, CDR-2, and/or CDR-3 as described. In some embodiments, the provided TCRs or antigen-binding fragments thereof (e.g., anti-HPV 16 E6 or anti-HPV 16 E7 TCRs) contain a Vβ region sequence, or a sufficient antigen-binding portion thereof, containing CDR-1, CDR-2, and/or CDR-3 as described. In some embodiments, a TCR or antigen-binding fragment thereof (e.g., an anti-HPV 16 E6 or anti-HPV 16 E7 TCR) contains a Vα region sequence containing CDR-1, CDR-2, and/or CDR-3 as described; and contains a Vβ region sequence containing CDR-1, CDR-2, and/or CDR-3 as described. The TCRs provided are also TCRs having sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.

In some embodiments, the TCR comprises _a Vα region comprising _a complementary determining region _{3 ( CDR - 3 )} comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13 (SEQ ID NO: ₂₉₂ ), wherein _X1 = A or G; _X2 = A, G, P, V _, or L; _X3 = A, K, N _, Y, D, or _S ; _X4 = R, M, I, T, or G; _X5 = N, E, L, D, V, or absent; _X6 = Y, G, N, D, or absent; _X7 = S, N, G, or absent; _X8 = G, _N , Y, or absent; _X9 = D, F, G, N, or absent; _X10 = A, N, Q, or Y; _X11 = K, R, or N; _X12 = F, L, or Y; _X13 =I, M, S, T, V, or Y. In some embodiments, the TCR comprises _{a Vα} region comprising _a complementary determining region _{3 ( CDR - 3} ) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13 (SEQ ID NO: 356), wherein _X1 = _A or G; _X2 =A, G, P _, V _or L; _X3 =A, K, N, Y, D or S; _X4 =R, M, I, T _{or G; X5=N, E, L, V or absent; X6 = Y} , G, N, D or absent; _X7 =S, N, G or absent; _X8 =G, N, Y or absent; _X9 =D, F, G, N or absent; _X10 =A, N, Q or Y; _X11 =K, R or N; _{X12 =} F, L or Y; _X13 = I, M, S, _T , _V or Y. In some embodiments, _the TCR contains _a Vα region containing _a complementary determining region 3 (CDR _{- 3} ) comprising the amino acid sequence _{AVNX4X5X6X7X8NX10X11LX13} (SEQ ID NO: 293), wherein _X4 = M or I; _X5 = E or L; _X6 = G or N; _X7 = S, G or absent; _X8 = S or N; _X10 = Y or A; _X11 = K or R; _X13 = T or M.

In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing a complementary determining region 3 (CDR-3) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 12, 50, 80, 106, 129, 150, 172, 198, 224, or 250, or a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences. In some aspects, the TCR or antigen-binding fragment thereof contains a _Vα region that contains a CDR3 contained within the following sequence: the amino acid sequence shown in any one of SEQ ID NO: 9, 47, 77, 103, 126, 149, 169, 195, 221 or 247, or a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such a sequence.

In some embodiments, the TCR contains a _Vβ region comprising _a complementary determining region _{3 ( CDR - 3} ) comprising the amino acid sequence _{X1X2X3X4X5X6X7X8X9X10X11X12X13} (SEQ ID NO: ₂₈₉ ), wherein _X1 = A _or S; _X2 = A, I, S or Y; _X3 = S, T _or R; _X4 = G, K, R, _L , P, S or Q; _X5 = D, G, R, T or W; _X6 = A, E, F, R, S, Q, T or V; _X7 = A, R, G, Y _or absent; _X8 = G, N, S or absent; _X9 = A, D, G, S or Y; _X10 = L, N or Y; _X11 = E or V; _X12 = L or Q; X13 = F, Y or T. In some embodiments, the TCR contains a _Vβ region comprising _a complementary determining region _{3 ( CDR - 3} ) comprising the amino acid sequence _{X1X2X3X4X5X6X7X8X9X10X11X12X13} (SEQ ID NO: 357), wherein _X1 = A _or S; _X2 = A, I, S or Y; _X3 = S, T _or R; _X4 = G, K, R, _L , P, S _or Q; _X5 = D, G, T or W; _X6 = A, E, F, R, S, Q, T or V; _X7 = A, R, G, Y _{or absent; X8 = G, N, S or absent; X9 = A} , D, G, S or Y; _X10 = L, N or Y; _X11 = E or V; _X12 = L or Q; X13 = F, Y or T. In some embodiments, the TCR contains a Vβ region comprising _a complementation determining region 3 ( _{CDR -} 3 ₎ comprising the amino acid sequence _{X1X2X3X4X5X6X7X8X9X10EQF} (SEQ ID NO: 290), wherein _X1 = _{A or} S; _X2 =A _or S; _X3 =S or R; _X4 =S, L or P; _X5 =W, D or R; _X6 =R, _T or Q; _X7 =R, G, Y or absent; _X8 =G, N or S; _X9 =G, D or Y; _{X10 =} N or L. In some embodiments, the TCR contains _a Vβ region comprising _a complementation determining region 3 ( _{CDR - 3 )} comprising the amino acid sequence _{X1X2X3X4X5X6X7X8X9X10EQY} (SEQ ID NO: 291), wherein _X1 =A _{or S} ; _X2 =I, S or Y; _X3 =S or T; _X4 =G, P, R or T; _X5 =R _or T; _X6 =F, S, A or V; _X7 =S or T.

In some cases, the TCR contains a Vβ region containing a complementary determining region 3 (CDR-3) comprising an amino acid sequence as shown in any one of SEQ ID NOs: 27, 63, 91, 115, 138, 158, 183, 209, 235, or 261, or a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences. In some embodiments, the TCR contains a Vβ region containing a CDR3 contained in the following sequence: an amino acid sequence as shown in any one of SEQ ID NOs: 24, 60, 88, 114, 137, 157, 180, 206, 232, or 258, or a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.

In some embodiments, the Vα region contains a complementary determining region 1 (CDR-1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 10, 48, 78, 104, 127, 170, 196, 222, or 248, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such sequences. In some aspects, the Vα region contains a CDR-1 contained within the following sequence: an amino acid sequence as set forth in any one of SEQ ID NOs: 9, 47, 77, 103, 126, 149, 169, 195, 221, or 247, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such sequences. In some embodiments, the Vα region contains a complementary determining region 2 (CDR-2) comprising an amino acid sequence as shown in any one of SEQ ID NOs: 11, 49, 79, 105, 128, 171, 197, 223, or 249, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such sequences. In some embodiments, the Vα region contains a CDR-2 contained within the following sequence: an amino acid sequence as shown in any one of SEQ ID NOs: 9, 47, 77, 103, 126, 149, 169, 195, 221, or 247, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such sequences.

In some cases, the Vβ region contains a complementary determining region 1 (CDR-1) comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 25, 61, 89, 181, 207, 233, or 259, or a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences. In some aspects, the Vβ region contains a CDR-1 contained within an amino acid sequence as set forth in any one of SEQ ID NOs: 24, 60, 88, 114, 137, 157, 180, 206, 232, or 258, or a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences. In some embodiments, the Vβ region contains a complementary determining region 2 (CDR-2) comprising an amino acid sequence as shown in any one of SEQ ID NOs: 26, 62, 90, 182, 208, 234, or 260, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such sequences. In some embodiments, the Vβ region contains a CDR-2 contained in the following sequence: An amino acid sequence as shown in any one of SEQ ID NOs: 24, 60, 88, 114, 137, 157, 180, 206, 232, or 258, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to such sequences.

In some embodiments, the Vα region contains the amino acid sequence set forth in any one of SEQ ID NOs: 9, 47, 77, 103, 126, 149, 169, 195, 221, or 247, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some cases, the Vβ region contains the amino acid sequence set forth in any one of SEQ ID NOs: 24, 60, 88, 114, 137, 157, 180, 206, 232, or 258, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the TCR contains an alpha chain that includes any of such Valpha chain sequences and any of such Vbeta chain sequences.

In some embodiments, the alpha chain of the TCR or antigen-binding fragment thereof further contains an alpha constitutive (Cα) region or a portion thereof. In some aspects, the beta chain further contains a beta constitutive (Cβ) region or a portion thereof. Thus, in some embodiments, a TCR, such as an HPV 16 E6 or E7 TCR or antigen-binding fragment thereof, contains an alpha chain comprising a variable alpha (Vα) region and an alpha constitutive (Cα) region or a portion thereof and/or a beta chain comprising a variable beta (Vβ) region and a beta constitutive (Cβ) region or a portion thereof.

In some cases, the Cα region and the Cβ region are mouse constant regions, such as native or variant (e.g., cysteine-substituted) mouse constant regions, including any mouse constant regions as described above. In some embodiments, the Cα region contains an amino acid sequence shown in SEQ ID NO: 15, 275, 276, 277, 278, 279, 281 or 282, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some cases, the Cβ region contains the amino acid sequence shown in SEQ ID NO: 30, 283, 284 or 285, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the Cα region contains the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the Cβ region contains the amino acid sequence shown in SEQ ID NO: 30.

In some embodiments, the Cα region and the Cβ region are human constant regions, such as natural or variant (e.g., cysteine-substituted) human constant regions, including any human constant regions as described above. In some such embodiments, the Cα region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 14, 333, 334, 335, 336, 337, 338, 341, 344, 345, 346, 347, or 348, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some aspects, the Cβ region contains an amino acid sequence as set forth in SEQ ID NO: 29, 339, 340, 342, 343, 349, 350, 351, or 352, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the Cα region contains an amino acid sequence as set forth in SEQ ID NO: 14. In some embodiments, the Cβ has or comprises an amino acid sequence as set forth in SEQ ID NO: 350. In some embodiments, the Cβ region contains an amino acid sequence as set forth in SEQ ID NO: 29.

Such Cα region and/or Cβ region are regions that have been modified, for example, by incorporation of one or more non-natural cysteine residues. In some embodiments, the modification corresponds to the introduction of cysteine at the following positions: at residue Thr48 of the Cα chain and/or residue Ser57 of the Cβ chain; at residue Thr45 of the Cα chain and/or residue Ser77 of the Cβ chain; at residue Tyr10 of the Cα chain and/or residue Ser17 of the Cβ chain; at residue Thr45 of the Cα chain and residue Asp59 of the Cβ chain; and/or at residue Ser15 of the Cα chain and residue Glu15 of the Cβ chain, with reference to the numbering of Cα shown in any one of SEQ ID NOs: 333-335 or 337 or Cβ shown in SEQ ID NOs: 339 or 340.

In some such embodiments, the Cα region contains a non-natural cysteine at the position corresponding to residue 48 and comprises the amino acid sequence set forth in any one of SEQ ID NO: 14, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and which contains one or more introduced non-natural cysteine residues. In some aspects, the Cβ region contains a non-natural cysteine at a position corresponding to residue 57 and contains the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and it contains one or more non-natural cysteine residues. In some aspects, the Cβ region contains a non-natural cysteine at a position corresponding to residue 57 and contains the amino acid sequence set forth in SEQ ID NO: 350, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and it contains one or more non-natural cysteine residues.

In some embodiments, the TCR or antigen-binding fragment thereof comprises an alpha chain that is or comprises the amino acid sequence set forth in SEQ ID NO: 5, 43, 73, 99, 122, 145, 165, 191, 217 or 243, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and/or a beta chain that is or comprises SEQ ID NO: The amino acid sequence shown in NO: 20, 56, 84, 110, 133, 153, 176, 202, 228 or 254, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the TCR or antigen-binding fragment thereof comprises an alpha chain that is or comprises the amino acid sequence shown in SEQ ID NO: 5, 43, 73, 99, 122, 145, 165, 191, 217 or 243, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and/or a beta chain that is or comprises SEQ ID NO: The amino acid sequence shown in NO: 369, 359, 370, 371, 372, 373, 374, 375, 376 or 377, or an amino acid sequence having at least 90% sequence identity therewith, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith.

In some such embodiments, the Cα region contains a non-natural cysteine at the position corresponding to residue 48 and comprises the amino acid sequence set forth in any one of SEQ ID NO: 15, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and which contains one or more introduced non-natural cysteine residues. In some embodiments, the Cβ region contains a non-natural cysteine at the position corresponding to residue 57 and contains the amino acid sequence set forth in SEQ ID NO: 30, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto and containing one or more non-natural cysteine residues.

In some embodiments, the TCR or antigen-binding fragment thereof comprises an alpha chain that is or comprises the amino acid sequence shown in SEQ ID NO: 6, 44, 74, 100, 123, 146, 166, 192, 218 or 244, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and/or a beta chain that is or comprises SEQ ID NO: The amino acid sequence shown in NO: 21, 57, 85, 111, 134, 154, 177, 203, 229 or 255, or an amino acid sequence having at least 90% sequence identity therewith, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith.

In some embodiments, the alpha chain and/or beta chain of the TCR is encoded by a nucleotide sequence comprising a signal peptide (also referred to as a leader sequence). Non-limiting examples of such signal peptides are signal peptides having or comprising an amino acid sequence as shown in any one of SEQ ID NOs: 13, 28, 51, 64, 81, 92, 107, 130, 173, 199, 225, 251, 184, 210, 236, 262, 286, 294, 327-332, 349, and 353-355.

In some embodiments, the TCR or antigen-binding fragment thereof has an alpha chain that is or comprises the amino acid sequence shown in SEQ ID NO: 7, 45, 75, 101, 124, 147, 167, 193, 219 or 245, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and/or a beta chain that is or comprises SEQ ID NO: The amino acid sequence shown in NO: 22, 58, 86, 112, 135, 155, 178, 204, 230 or 256, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the TCR or antigen-binding fragment thereof is encoded by a nucleotide sequence that encodes: a) an alpha chain that is or comprises the amino acid sequence shown in SEQ ID NO: 7, 45, 75, 101, 124, 147, 167, 193, 219 or 245, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and/or b) a beta chain that is or comprises SEQ ID NO: The amino acid sequence shown in NO: 22, 58, 86, 112, 135, 155, 178, 204, 230 or 256, or an amino acid sequence having at least 90% sequence identity therewith, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith.

In some embodiments, the TCR or antigen-binding fragment thereof has an alpha chain that is or comprises the amino acid sequence shown in SEQ ID NO: 7, 45, 75, 101, 124, 147, 167, 193, 219 or 245, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and/or a beta chain that is or comprises SEQ ID NO: The amino acid sequence shown in NO:378, 360, 379, 380, 381, 382, 383, 384, 385 or 386, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the TCR or antigen-binding fragment thereof is encoded by a nucleotide sequence that encodes: a) an alpha chain that is or comprises the amino acid sequence shown in SEQ ID NO: 7, 45, 75, 101, 124, 147, 167, 193, 219 or 245, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and/or b) a beta chain that is or comprises SEQ ID NO: The amino acid sequence shown in NO: 378, 360, 379, 380, 381, 382, 383, 384, 385 or 386, or an amino acid sequence having at least 90% sequence identity therewith, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith.

In some embodiments, the nucleic acid encoding the alpha strand and the nucleic acid encoding the beta strand can be linked via a linker, such as any linker described elsewhere herein.

In some embodiments, the TCR or antigen-binding fragment thereof has an alpha chain that is or comprises the amino acid sequence shown in SEQ ID NO: 8, 46, 76, 102, 125, 148, 168, 194, 220 or 246, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and/or a beta chain that is or comprises SEQ ID NO: The invention relates to an amino acid sequence as set forth in NO: 23, 59, 87, 113, 136, 156, 179, 205, 231 or 257, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the TCR or antigen-binding fragment thereof is encoded by a nucleotide sequence that encodes: a) an alpha chain that is or comprises the amino acid sequence shown in SEQ ID NO: 8, 46, 76, 102, 125, 148, 168, 194, 220 or 246, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and/or b) a beta chain that is or comprises SEQ ID NO: NO: 23, 59, 87, 113, 136, 156, 179, 205, 231 or 257, or an amino acid sequence having at least 90% sequence identity therewith, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity therewith. In some embodiments, the nucleic acid encoding the alpha chain and the nucleic acid encoding the beta chain can be linked via a linker, such as any linker described elsewhere herein.

In some embodiments, the TCR or antigen-binding fragment thereof provided herein is a TCR or antigen-binding fragment thereof that binds to or recognizes an antigen expressed on the surface of a cancer cell and/or a cell infected with HPV or containing an HPV DNA sequence. In some aspects, such binding or recognition is related to a cell line named SCC152. In some aspects, an engineered T cell containing or expressing such a TCR or antigen-binding fragment thereof exhibits cytotoxic activity when in contact with a cancer target cell and/or a target cell infected with HPV or containing an HPV DNA sequence (e.g., a SCC152 cell).

Examples of such TCRs or antigen-binding fragments are those shown in Tables 2A and 2B , as shown in the columns therein. In some embodiments, the Vα region and the Vβ region contain amino acid sequences corresponding to SEQ ID NO: in Tables 2A or 2B , as shown in the columns therein. In some embodiments, the Vα region and the Vβ region contain CDR-1, CDR-2, and CDR-3 sequences contained in the Vα region and the Vβ region in Tables 2A or 2B , as shown in the columns therein. In some aspects, the TCR contains constant α and constant β region sequences, such as those sequences corresponding to SEQ ID NO: in Tables 2A or 2B , as shown in the columns therein. In some cases, the TCR contains a full sequence comprising variable and constant chains, such as a sequence corresponding to SEQ ID NO: in Table 2A or 2B ("full"), such as shown in each column therein. In some embodiments, the full sequence containing the variable region and the constant region also includes a signal sequence and thus contains a sequence corresponding to SEQ ID NO: in Table 2A or 2B ("full + signal"), such as shown in each column therein. The TCR provided is also a TCR containing a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such a sequence. Exemplary TCRs containing such sequences or modified versions thereof as described elsewhere herein are also shown in Tables 2A and 2B , such as in each column therein, respectively.

In some embodiments, the TCR or antigen binding fragment thereof provided herein is a TCR or antigen binding fragment thereof that recognizes or binds to an antigenic determinant or region of HPV16 E6, such as a TCR or antigen binding fragment thereof that comprises a peptide antigenic determinant E6 (29-38) of the amino acid sequence TIHDIILECV (SEQ ID NO.268). In some aspects, the TCR or antigen binding fragment thereof provided herein is a TCR or antigen binding fragment thereof that recognizes or binds to an antigenic determinant or region of the HPV16 E7 protein, such as a TCR or antigen binding fragment thereof that comprises a peptide antigenic determinant E7 (11-19) of the amino acid sequence YMLDLQPET (SEQ ID NO.271). Examples of such TCRs are described below.

a. HPV 16 E6 (29-38)

Anti-HPV 16 E6 (29-38) TCRs or antigen-binding fragments thereof are provided herein. In some cases, the TCR recognizes or binds to a peptide antigenic determinant derived from HPV16 E6, which is or contains E6 (29-38) TIHDIILECV (SEQ ID NO: 268). In some embodiments, the TCR recognizes or binds to HPV 16 E6 (29-38) in the context of an MHC, such as MHC class I, such as HLA-A2. In some embodiments, the provided TCR or antigen-binding fragment thereof is capable of or binds to an HPV 16 E6 (29-38)-peptide-MHC tetramer complex.

In some embodiments, the Vα region contains a complementation determining region 3 (CDR-3) comprising the amino acid sequence set forth in SEQ ID NO: 250, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the Vα region further contains a complementation determining region 1 (CDR-1) comprising the amino acid sequence set forth in SEQ ID NO: 248, or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the Vα region further contains a complementary determining region 2 (CDR-2) comprising an amino acid sequence as shown in SEQ ID NO: 249 or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with such a sequence. In some cases, the Vα region contains CDR-1, CDR-2 and CDR-3 contained in the following sequence: an amino acid sequence as shown in SEQ ID NO: 247 or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with such a sequence.

In some embodiments, the Vβ region contains a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 261 or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with such a sequence. In some embodiments, the Vβ region further contains a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 259 or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with such a sequence. In some embodiments, the Vβ region further contains a complementary determining region 2 (CDR-2) comprising an amino acid sequence as shown in SEQ ID NO: 260 or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with such a sequence. In some examples, the Vβ region contains CDR-1, CDR-2 and CDR-3 contained in the following sequence: an amino acid sequence as shown in SEQ ID NO: 258 or a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with such a sequence.

In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region comprising: a complementary determining region 1 (CDR-1) comprising the amino acid sequence set forth in SEQ ID NO: 248; a complementary determining region 2 (CDR-2) comprising the amino acid sequence set forth in SEQ ID NO: 249; and/or a complementary determining region 3 (CDR-3) comprising the amino acid sequence set forth in SEQ ID NO: 250. The TCRs provided are also TCRs having sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences. In some embodiments, the TCR or antigen-binding fragment thereof contains a Vβ region comprising: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 259; a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 260; and/or a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 261. The TCR provided is also a TCR having a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such a sequence.

In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region, which contains CDR-1, CDR-2 and CDR-3, which contain the amino acid sequences of SEQ ID NOs: 248, 249 and 250, respectively; and a Vβ region, which contains CDR-1, CDR-2 and CDR-3, which contain the amino acid sequences of SEQ ID NOs: 259, 260 and 261, respectively. The TCR provided is also a TCR having a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences.

In some embodiments, the TCR or antigen-binding fragment includes a Vα region containing a complementary determining region 1 (CDR-1), CDR-2, and CDR-3, which respectively include the CDR-1, CDR-2, and CDR-3 amino acid sequences shown in Table 3 ; and a Vβ region containing a complementary determining region 1 (CDR-1), CDR-2, and CDR-3, which respectively include the CDR-1, CDR-2, and CDR-3 amino acid sequences shown in Table 3. The TCRs provided are also TCRs containing sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences. Exemplary TCRs containing such CDRs or modified versions thereof as described elsewhere herein are also shown in Table 3 .

In some cases, the TCR or antigen-binding fragment thereof contains a Vα region and a Vβ region, which contain the amino acid sequences of SEQ ID NO: 247 and 258, respectively. The TCR provided is also a TCR containing a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences.

In some embodiments, the α chain of the TCR or antigen-binding fragment thereof further contains a Cα region or a portion thereof and/or the β chain further contains a Cβ region or a portion thereof. In some embodiments, the Cα region or a portion thereof is or comprises a human Cα region, such as a native human Cα region or a variant thereof, such as any one of SEQ ID NOs: 14, 333, 334, 335, 336, 337, 338, 341, 344, 345, 346, 347 or 348, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some aspects, the Cβ region or a portion thereof is or comprises a human Cβ region, such as a native human Cβ region or a variant thereof, such as shown in SEQ ID NO: 29, 339, 340, 342, 343, 349, 350, 351 or 352, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the Cα region and/or Cβ region is a variant modified, such as by incorporating one or more non-natural cysteine residues, such as any non-natural cysteine residues described herein. In some embodiments, the Cα region or a portion thereof contains a non-natural cysteine at residue 48 and comprises the amino acid sequence set forth in any one of SEQ ID NO: 14, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and it contains an introduced non-natural cysteine residue (e.g., Cys48). In some aspects, the Cβ region contains a non-natural cysteine at residue 57 and comprises the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some aspects, the Cβ region contains a non-native cysteine at residue 57 and contains the amino acid sequence set forth in SEQ ID NO: 350, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

In some embodiments, the TCR or antigen-binding fragment thereof comprises an alpha chain comprising the amino acid sequence shown in SEQ ID NO: 243 or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and/or a beta chain comprising the amino acid sequence shown in SEQ ID NO: 254 or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

In some embodiments, the TCR or antigen-binding fragment thereof comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO: 243 or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and/or a beta chain comprising the amino acid sequence set forth in SEQ ID NO: 377 or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

In some embodiments, the Cα region or a portion thereof is or comprises a mouse Cα region, such as a native mouse Cα region or a variant thereof, such as shown in any one of SEQ ID NOs: 15, 275, 276, 277, 278, 279, 281 or 282, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some aspects, the Cβ region or a portion thereof is or comprises a mouse Cβ region, such as a native mouse Cβ region or a variant thereof, such as shown in SEQ ID NO: 30, 283, 284 or 285, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the Cα region and/or Cβ region is a variant modified, such as by incorporating one or more non-natural cysteine residues, such as any of the non-natural cysteine residues described herein. In some embodiments, the Cα region or a portion thereof contains a non-natural cysteine at residue 48 and comprises an amino acid sequence as set forth in any one of SEQ ID NO: 15, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and it contains an introduced non-natural cysteine residue (e.g., Cys48). In some aspects, the Cβ region contains a non-natural cysteine at residue 57 and comprises an amino acid sequence as set forth in SEQ ID NO: 30, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

In some embodiments, the TCR or antigen-binding fragment thereof comprises an alpha chain comprising the amino acid sequence shown in SEQ ID NO: 244 or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and/or a beta chain comprising the amino acid sequence shown in SEQ ID NO: 255 or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

Exemplary TCRs or antigen-binding fragments include those shown in Tables 4A and 4B , as shown in the respective columns thereof. In some embodiments, the Vα region and the Vβ region contain amino acid sequences corresponding to SEQ ID NO: in Tables 4A or 4B , as shown in the respective columns thereof. In some embodiments, the Vα region and the Vβ region contain CDR-1, CDR-2, and CDR-3 sequences contained in the Vα region and the Vβ region in Tables 4A or 4B , as shown in the respective columns thereof. In some aspects, the TCR contains constant α and constant β region sequences, such as those sequences corresponding to SEQ ID NO: in Tables 4A or 4B , as shown in the respective columns thereof. In some cases, the TCR contains a full sequence comprising variable and constant chains, such as the sequence corresponding to SEQ ID NO: in Table 4A or 4B ("full"), such as shown in each column therein. In some embodiments, the full sequence containing the variable and constant regions also includes a signal sequence and thus contains a sequence corresponding to SEQ ID NO: in Table 4A or 4B ("full + signal"), such as shown in each column therein. The TCR provided is also a TCR containing a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such a sequence. Exemplary TCRs containing such sequences or modified versions thereof as described elsewhere herein are also shown in Tables 4A and 4B , such as in each column therein, respectively.

b. HPV 16 E7 (11-19)

Anti-HPV 16 E7 (11-19) TCRs or antigen-binding fragments thereof are provided herein. In some cases, the TCR recognizes or binds to a peptide antigenic determinant derived from HPV 16 E7 that is or contains E7 (11-19) YMLDLQPET (SEQ ID NO: 271). In some embodiments, the TCR recognizes or binds to HPV 16 E7 (11-19) in the context of an MHC, such as MHC class I, e.g., HLA-A2. In some embodiments, the provided TCRs or antigen-binding fragments thereof are capable of or bind to an HPV 16 E7 (11-19)-peptide-MHC tetramer complex. In some embodiments, engineered T cells containing or expressing such TCRs or antigen-binding fragments thereof exhibit cytotoxic activity when contacted with cancer target cells and/or target cells infected with HPV or containing HPV DNA sequences (e.g., SCC152 cells).

In some embodiments, the TCR or antigen-binding fragment _thereof comprises _{a Vα} region comprising _a complementary _determining region 3 (CDR- ₃ ) comprising the amino acid sequence _{X1X2X3X4X5X6X7X8X9X10X11X12X13} (SEQ ID NO: 292), wherein _X1 = A or G; _X2 = A, G, P _, V _or L; _X3 = A, K, N _{, Y} , D or S _{; X4} = R, M, I, T or G; _X5 = N, E, L, D, V or absent; _X6 = Y, G, N, D or absent; _X7 = S, N, G or absent; _X8 = G, N, Y or absent; _X9 = D, F, G, N or absent; _X10 = A, N, Q or Y; _X11 = K, R or N; _{X12 =} =F, L or Y; and X ₁₃ =I, M, S, T, V or Y. In some embodiments, the TCR comprises _{a Vα} region comprising _a complementary determining region _{3 ( CDR - 3} ) comprising the amino acid sequence X1X2X3X4X5X6X7X8X9X10X11X12X13 (SEQ ID NO: 356), wherein _X1 = _A or G; _X2 =A, G, P _, V _or L; _X3 =A, K, N, Y, D or S; _X4 =R, M, I, T _{or G; X5=N, E, L, V or absent; X6 = Y} , G, N, D or absent; _X7 =S, N, G or absent; _X8 =G, N, Y or absent; _X9 =D, F, G, N or absent; _X10 =A, N, Q or Y; _X11 =K, R or N; _{X12 =} F, L or Y; _X13 =I, M, S, T, V, or Y.

In some embodiments _, the TCR or _antigen -binding fragment _thereof contains a _Vα region containing a complementary determining region 3 ( _CDR -3) comprising the amino acid sequence AVNX4X5X6X7X8X9X10X11LX13 (SEQ ID NO: 293), _X4 = M _or I; _X5 = E or L; _X6 = G or _N ; _X7 = S, G or absent; _X8 = S or N; _X10 = Y or A; _X11 = _K or R; and _X13 = _T or M.

In some aspects, the TCR or antigen-binding fragment _thereof contains a _Vβ region comprising a complementary determining region 3 (CDR _- 3) comprising the amino acid sequence _{X1X2X3X4X5X6X7X8X9X10X11X12X13} (SEQ ID NO: 289), wherein _X1 = A or S _{; X2 =} A, I, S or Y; _X3 = S, T or R; X4 = G, K, R, _{L , P} , S _or Q; X5 = D, G, R, T or W; _X6 = A, E, F, R, S, Q, T or V; X7 = A, R, G, Y or absent; _X8 = G, N, S or absent; _X9 = A, D, G, S or Y; _X10 = L, N or Y; X11 = E or V; X12 = A, R, G, Y or absent; _X13 = A, D, G, S or Y; _X14 = L, N or Y; X15 = E or V; X16 = A, R, G, Y or absent; _X17 = A, R, G, Y or absent; _X18 = G, N, S or absent; _X19 = A, D, G, S or Y; X20 = L, N or Y; _X21 = E or V; X22 = A, I, S or Y; X23 = S, T or R; X24 = G, K, R, L, P, S or Q; X25 = D, G, R, T or W; _X26 = A, I, S or Y; X27 = L, N or Y; _X28 = E or V; =L or Q; X13=F, Y or T. In some embodiments, the TCR contains a _Vβ region comprising _a complementary determining region _{3 ( CDR - 3} ) comprising the amino acid sequence _{X1X2X3X4X5X6X7X8X9X10X11X12X13} (SEQ ID NO: 357), wherein _X1 = A _or S; _X2 = A, I, S or Y; _X3 = S, T _or R; _X4 = G, K, R, _L , P, S _or Q; _X5 = D, G, T or W; _X6 = A, E, F, R, S, Q, T or V; _X7 = A, R, G, Y _{or absent; X8 = G, N, S or absent; X9 = A} , D, G, S or Y; _X10 = L, N or Y; _X11 = E or V; _X12 = L or Q; X13 = F, Y or T.

In some embodiments, the TCR or antigen-binding fragment _thereof contains a _Vβ region comprising _a complementary determining region 3 ₍ CDR-3) comprising the amino acid sequence _{X1X2X3X4X5X6X7X8X9X10EQF} (SEQ ID NO: 290), wherein _X1 =A _or S; _X2 =A or S; _X3 =S or _R ; _X4 =S, L or P; _X5 =W, D or R _{; X6} =R, T or Q; _X7 =R, G, Y or absent; _X8 =G, _N or S; _{X9 =} G, D or Y; _X10 =N or L.

In some embodiments, the TCR or antigen-binding fragment _thereof contains _{a Vβ} region comprising _a complementary determining region 3 ₍ CDR-3) comprising the amino acid sequence _{X1X2X3X4X5X6X7X8X9X10EQY} (SEQ ID NO: 291), wherein _X1 =A _or S; _X2 =I, S or Y; _{X3 =} S or T; _X4 =G, P _, R or T; _X5 =R or T; _X6 = _F , S, A or V; _X7 =S or T.

In some embodiments, the Vα region contains: a complementary determining region 3 (CDR-3) comprising an amino acid sequence as shown in any one of SEQ ID NOs: 50, 80, 106, 129, 150, 172, 198, or 224; or a CDR3 contained within an amino acid sequence as shown in any one of SEQ ID NOs: 47, 77, 103, 126, 149, 169, 195, or 221. In some embodiments, the Vα region contains a CDR3 sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.

In some embodiments, the Vα region further contains: a complementary determining region 1 (CDR-1) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 48, 78, 104, 127, 170, 196, or 222; or a CDR1 contained within the amino acid sequence set forth in any one of SEQ ID NOs: 47, 77, 103, 126, 149, 169, 195, or 221. In some embodiments, the Vα region contains a CDR-1 sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences. In some aspects, the Vα region further contains: a complementary determining region 2 (CDR-2) comprising an amino acid sequence as shown in any one of SEQ ID NOs: 49, 79, 105, 128, 171, 197, or 223; or a CDR2 contained within an amino acid sequence as shown in any one of SEQ ID NOs: 47, 77, 103, 126, 149, 169, 195, or 221. In some embodiments, the Vα region contains a CDR-1 sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.

In some aspects, the Vβ region contains: a complementary determining region 3 (CDR-3) comprising an amino acid sequence as shown in any one of SEQ ID NOs: 27, 63, 91, 115, 138, 158, 183, 209, or 235; or a CDR3 contained within an amino acid sequence as shown in any one of SEQ ID NOs: 60, 88, 114, 137, 157, 180, 206, or 232. In some embodiments, the Vβ region contains a CDR3 sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.

In some embodiments, the Vβ region contains: a complementary determining region 1 (CDR-1) comprising the amino acid sequence set forth in SEQ ID NO: 61, 89, 181, 207, or 233; or a CDR-1 contained within the amino acid sequence set forth in any one of SEQ ID NO: 60, 88, 114, 137, 157, 180, 206, or 232. In some embodiments, the Vβ region contains a CDR-1 sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences. In some cases, the Vβ region contains: a complementary determining region 2 (CDR-2) comprising an amino acid sequence shown in SEQ ID NO: 62, 90, 182, 208, or 234; or a CDR-2 contained within an amino acid sequence shown in any one of SEQ ID NO: 60, 88, 114, 137, 157, 180, 206, or 232. In some embodiments, the Vβ region contains a CDR-2 sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences.

In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing CDR-1, CDR-2 and CDR-3, which respectively contain the amino acid sequences of SEQ ID NOs: 48, 49 and 50; and a Vβ region containing CDR-1, CDR-2 and CDR-3, which respectively contain the amino acid sequences of SEQ ID NOs: 61, 62 and 63. The TCR provided is also a TCR having a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences.

In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing CDR-1, CDR-2 and CDR-3, which contain amino acid sequences of SEQ ID NOs: 78, 79 and 80, respectively; and a Vβ region containing CDR-1, CDR-2 and CDR-3, which contain amino acid sequences of SEQ ID NOs: 89, 90 and 91, respectively. The TCR provided is also a TCR having a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences.

In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing CDR-1, CDR-2 and CDR-3, which respectively contain the amino acid sequences of SEQ ID NOs: 104, 105 and 106; and a Vβ region containing CDR-1, CDR-2 and CDR-3, which respectively contain the amino acid sequences of SEQ ID NOs: 89, 90 and 115. The TCR provided is also a TCR having a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences.

In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing CDR-1, CDR-2 and CDR-3, which respectively contain the amino acid sequences of SEQ ID NOs: 127, 128 and 129; and a Vβ region containing CDR-1, CDR-2 and CDR-3, which respectively contain the amino acid sequences of SEQ ID NOs: 89, 90 and 138. The TCR provided is also a TCR having a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences.

In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing CDR-1, CDR-2 and CDR-3, which respectively contain the amino acid sequences of SEQ ID NOs: 127, 128 and 150; and a Vβ region containing CDR-1, CDR-2 and CDR-3, which respectively contain the amino acid sequences of SEQ ID NOs: 89, 90 and 158. The TCR provided is also a TCR having a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences.

In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region, which contains CDR-1, CDR-2 and CDR-3, which respectively contain the amino acid sequences of SEQ ID NOs: 170, 171 and 172; and a Vβ region, which contains CDR-1, CDR-2 and CDR-3, which respectively contain the amino acid sequences of SEQ ID NOs: 181, 182 and 183. The TCR provided is also a TCR having a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences.

In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing CDR-1, CDR-2 and CDR-3, which respectively contain the amino acid sequences of SEQ ID NOs: 196, 197 and 198; and a Vβ region containing CDR-1, CDR-2 and CDR-3, which respectively contain the amino acid sequences of SEQ ID NOs: 207, 208 and 209. The TCR provided is also a TCR having a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences.

In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region containing CDR-1, CDR-2 and CDR-3, which respectively contain the amino acid sequences of SEQ ID NOs: 222, 223 and 224; and a Vβ region containing CDR-1, CDR-2 and CDR-3, which respectively contain the amino acid sequences of SEQ ID NOs: 233, 234 and 235. The TCR provided is also a TCR having a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences.

In some cases, the Vα region contains a complementary determining region 1 (CDR-1), CDR-2, and CDR-3, which respectively comprise the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within the Vα region amino acid sequence shown in any one of SEQ ID NOs: 47, 77, 103, 126, 149, 169, 195, or 221. In some cases, the Vβ region contains a complementary determining region 1 (CDR-1), CDR-2, and CDR-3, which respectively comprise the CDR-1, CDR-2, and CDR-3 amino acid sequences contained within the Vβ region amino acid sequence shown in any one of SEQ ID NOs: 60, 88, 114, 137, 157, 180, 206, or 232. The TCRs provided are also TCRs containing sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences.

In some embodiments, the TCR or antigen-binding fragment includes a Vα region containing a complementary determining region 1 (CDR-1), CDR-2, and CDR-3, which respectively include the CDR-1, CDR-2, and CDR-3 amino acid sequences shown in Table 5 ; and a Vβ region containing a complementary determining region 1 (CDR-1), CDR-2, and CDR-3, which respectively include the CDR-1, CDR-2, and CDR-3 amino acid sequences shown in Table 5. The TCRs provided are also TCRs containing sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences. Exemplary TCRs containing such CDRs or modified versions thereof as described elsewhere herein are also shown in Table 5 .

In some embodiments, the TCR or antigen-binding fragment thereof contains a Vα region and a Vβ region, which contain the amino acid sequences of SEQ ID NOs: 47 and 60, respectively. In some aspects, the Vα region and the Vβ region contain the amino acid sequences of SEQ ID NOs: 77 and 88, respectively. In some aspects, the Vα region and the Vβ region contain the amino acid sequences of SEQ ID NOs: 103 and 114, respectively. In some cases, the Vα region and the Vβ region contain the amino acid sequences of SEQ ID NOs: 126 and 137, respectively. In some cases, the Vα region and the Vβ region contain the amino acid sequences of SEQ ID NOs: 149 and 157, respectively. In some aspects, the Vα region and the Vβ region contain the amino acid sequences of SEQ ID NOs: 169 and 180, respectively. In some embodiments, the Vα region and the Vβ region contain the amino acid sequences of SEQ ID NOs: 195 and 206, respectively. In some cases, the Vα region and the Vβ region contain the amino acid sequences of SEQ ID NO: 221 and 232, respectively. The TCR provided is also a TCR containing a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences.

In some embodiments, the α chain of the TCR or antigen-binding fragment thereof further contains a Cα region or a portion thereof and/or the β chain further contains a Cβ region or a portion thereof. In some embodiments, the Cα region or a portion thereof is or comprises a human Cα region, such as a native human Cα region or a variant thereof, such as any one of SEQ ID NOs: 14, 333, 334, 335, 336, 337, 338, 341, 344, 345, 346, 347, or 348, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some aspects, the Cβ region or a portion thereof is or comprises a human Cβ region, such as a native human Cβ region or a variant thereof, such as shown in SEQ ID NO: 29, 339, 340, 342, 343, 349, 350, 351 or 352, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the Cα region and/or Cβ region is a variant modified, such as by incorporating one or more non-natural cysteine residues, such as any non-natural cysteine residues described herein. In some embodiments, the Cα region or a portion thereof contains a non-natural cysteine at residue 48 and comprises an amino acid sequence as set forth in any one of SEQ ID NO: 14, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and it contains an introduced non-natural cysteine residue (e.g., Cys48). In some aspects, the Cβ region contains a non-natural cysteine at residue 57 and comprises an amino acid sequence as set forth in SEQ ID NO: 29, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some aspects, the Cβ region contains a non-native cysteine at residue 57 and contains the amino acid sequence set forth in SEQ ID NO: 350, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

In some embodiments, the TCR or antigen-binding fragment thereof comprises an alpha chain comprising the amino acid sequence set forth in SEQ ID NO:43, 73, 99, 122, 145, 165, 191 or 217, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and/or a beta chain comprising the amino acid sequence set forth in SEQ ID NO:56, 84, 110, 133153, 176, 202, 228 or 359, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the TCR or antigen-binding fragment thereof comprises an alpha chain comprising an amino acid sequence as set forth in SEQ ID NO: 43, 73, 99, 122, 145, 165, 191 or 217, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and/or a beta chain comprising an amino acid sequence as set forth in SEQ ID NO: 359, 370, 371, 372, 373, 374, 375 or 376, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

In some embodiments, the Cα region or a portion thereof is or comprises a mouse Cα region, such as a native mouse Cα region or a variant thereof, such as shown in any one of SEQ ID NOs: 15, 275, 276, 277, 278, 279, 281 or 282, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some aspects, the Cβ region or a portion thereof is or comprises a mouse Cβ region, such as a native mouse Cβ region or a variant thereof, such as shown in SEQ ID NO: 30, 283, 284 or 285, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the Cα region and/or Cβ region is a variant modified, such as by incorporating one or more non-natural cysteine residues, such as any non-natural cysteine residue described herein. In some embodiments, the Cα region or a portion thereof contains a non-natural cysteine at residue 48 and comprises an amino acid sequence as set forth in any one of SEQ ID NO: 15, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and it contains an introduced non-natural cysteine residue (e.g., Cys48). In some aspects, the Cβ region contains a non-natural cysteine at residue 57 and comprises an amino acid sequence as set forth in SEQ ID NO: 30, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

In some embodiments, the TCR or antigen-binding fragment thereof comprises an alpha chain comprising an amino acid sequence as set forth in SEQ ID NO: 46, 76, 102, 125, 148, 168, 194 or 220, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and/or a beta chain comprising an amino acid sequence as set forth in SEQ ID NO: 57, 85, 111, 134, 154, 177, 203 or 229, or an amino acid sequence having at least 90% sequence identity thereto, such as a sequence having at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

Exemplary TCRs or antigen-binding fragments include those shown in Tables 6A and 6B , as shown in the columns therein. In some embodiments, the Vα region and the Vβ region contain amino acid sequences corresponding to SEQ ID NO: in Table 6A or 6B , as shown in the columns therein. In some embodiments, the Vα region and the Vβ region contain CDR-1, CDR-2, and CDR-3 sequences contained in the Vα region and the Vβ region in Table 6A or 6B , as shown in the columns therein. In some aspects, the TCR contains constant α and constant β region sequences, such as those corresponding to SEQ ID NO: in Table 6A or 6B , as shown in the columns therein. In some cases, the TCR contains a full sequence comprising variable and constant chains, such as the sequence corresponding to SEQ ID NO: in Table 6A or 6B ("full"), such as shown in each column therein. In some embodiments, the full sequence containing the variable and constant regions also includes a signal sequence and thus contains a sequence corresponding to SEQ ID NO: in Table 6A or 6B ("full + signal"), such as shown in each column therein. The TCR provided is also a TCR containing a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such a sequence. Exemplary TCRs containing such sequences or modified versions thereof as described elsewhere herein are also shown in Tables 6A and 6B , such as in each column therein, respectively.

The TCR provided herein is a TCR containing Vα and Vβ, Vα having CDR1, 2 and 3 as shown in SEQ ID NOs: 48, 49 and 50, respectively, and Vβ having CDR1, 2 and 3 as shown in SEQ ID NOs: 61, 62 and 63, respectively. In some embodiments, such TCR contains Vα as shown in SEQ ID NO: 47 and Vβ as shown in SEQ ID NO: 60. In some embodiments, such TCR contains α chain and β chain, α chain further contains Cα region, and β chain further contains Cβ region. In some embodiments, Cα and Cβ are human constant regions or functional variants thereof. In some embodiments, such TCR contains α chain, α chain contains Cα human constant region or variant thereof, which contains non-natural cysteine substitutions, such as any non-natural cysteine substitutions described herein. In some embodiments, such TCRs contain an alpha chain containing a Calpha constant region as shown in SEQ ID NO: 14. In some embodiments, such TCRs contain a beta chain containing a Cbeta human constant region or a variant thereof containing a non-natural cysteine substitution, such as any non-natural cysteine substitution described herein. In some embodiments, such TCRs contain a beta chain containing a Cbeta region as shown in SEQ ID NO: 29 or SEQ ID NO: 350. In some embodiments, such TCRs contain Calpha as shown in SEQ ID NO: 14 and Cbeta as shown in SEQ ID NO: 29. In some embodiments, such TCRs contain Calpha as shown in SEQ ID NO: 14 and Cbeta as shown in SEQ ID NO: 350. In some embodiments, such TCRs contain an alpha chain as shown in SEQ ID NO: 43 and a beta chain as shown in SEQ ID NO: 56. In some embodiments, such TCRs contain an alpha chain as shown in SEQ ID NO: 43 and a beta chain as shown in SEQ ID NO: 359. In some embodiments, the TCR is encoded by a polynucleotide encoding a sequence as shown in SEQ ID NO: 69. In some embodiments, the TCR is encoded by a polynucleotide encoding a sequence as shown in SEQ ID NO: 362. The TCRs provided are also TCRs containing sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences, which in some aspects include Vα and Vβ, Vα having CDR1, 2 and 3 as shown in SEQ ID NO: 48, 49 and 50, respectively, and Vβ having CDR1, 2 and 3 as shown in SEQ ID NO: 61, 62 and 63, respectively.

2. Mutants and Modifications

In some embodiments, a binding molecule, such as a TCR or an antigen-binding fragment thereof, is modified or has been modified. In certain embodiments, the binding molecules, such as a TCR or an antigen-binding fragment thereof, comprise one or more amino acid changes, such as substitutions, deletions, insertions and/or mutations, compared to the sequence of the binding molecules (e.g., TCRs) described herein. Exemplary variants include variants designed to improve the binding affinity and/or other biological properties of the binding molecule. Amino acid sequence variants of the binding molecule can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the binding molecule or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the binding molecule. Any combination of deletions, insertions and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

In some embodiments, directed evolution is used to generate TCRs with altered properties, such as higher affinity for a particular peptide in the context of an MHC molecule. In some embodiments, directed evolution is achieved by display methods, including but not limited to yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54) or T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84). In some embodiments, the display method includes engineering or modifying a known parent or reference TCR. For example, in some cases, a reference TCR, such as any TCR provided herein, can be used as a template to generate mutation-induced TCRs with one or more residues of the CDR mutated, and mutants are selected that have the desired altered properties, such as higher affinity for a peptide epitope in the context of an MHC molecule.

In certain embodiments, a binding molecule, such as a TCR or antigen-binding fragment thereof, comprises one or more amino acid substitutions, such as compared to a binding molecule (e.g., TCR) sequence described herein and/or compared to a sequence of a natural repertoire, such as a human repertoire. Substitutional Sites of interest for mutation induction include CDRs, FRs, and/or constant regions. Amino acid substitutions can be introduced into a binding molecule of interest and the products screened for desired activities, such as retained/improved antigen affinity or avidity, reduced immunogenicity, improved half-life, CD8-independent binding or activity, surface expression, promotion of TCR chain pairing, and/or other improved properties or functions.

In some embodiments, one or more residues within a CDR of a parent binding molecule (e.g., a TCR) are substituted. In some embodiments, the substitution is made to restore the sequence or position in the sequence to a germline sequence, such as a binding molecule sequence found in the germline (e.g., the human germline), for example, to reduce the potential for immunogenicity, such as when administered to a human subject.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, as long as such changes do not substantially reduce the ability of the binding molecule (e.g., a TCR or antigen-binding fragment thereof) to bind to an antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) may be made in a CDR that do not substantially reduce binding affinity. Such changes may be made, for example, outside of the residues in the CDR that contact the antigen. In certain embodiments of the variable sequences provided herein, each CDR is unchanged or contains no more than one, two, or three amino acid substitutions.

Amino acid sequence insertions include amino-terminal and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as sequence-internal insertions of single or multiple amino acid residues.

In some embodiments, a TCR or an antigen-binding fragment thereof may contain one or more modifications in the α chain and/or the β chain, such that when the TCR or an antigen-binding fragment thereof is expressed in a cell, the frequency of mispairing between the TCR α chain and the β chain and the endogenous TCR α chain and the β chain is reduced, the expression of the TCR α chain and the β chain is increased, and/or the stability of the TCR α chain and the β chain is increased.

In some embodiments, the TCR contains one or more non-natural cysteine residues to introduce a covalent disulfide bond that connects the residues of the immunoglobulin region of the constant domain of the α chain to the residues of the immunoglobulin region of the constant domain of the β chain. In some embodiments, one or more cysteines can be incorporated into the extracellular sequence of the constant region of the first and second segments of the TCR polypeptide. Exemplary non-limiting modifications for introducing non-natural cysteine residues in TCR are described herein (see also International PCT Nos. WO2006/000830 and WO2006037960). In some cases, both natural and non-natural disulfide bonds are desired. In some embodiments, the TCR or antigen-binding fragment is modified such that the interchain disulfide bonds present in the native TCR are absent.

In some embodiments, the transmembrane domain of the constant region of the TCR can be modified to contain a larger number of hydrophobic residues (see, e.g., Haga-Friedman et al. (2012) Journal of Immunology, 188: 5538-5546). In some embodiments, the transmembrane region of the TCR α chain contains one or more mutations corresponding to S116L, G119V or F120L, with reference to the number of Cα shown in any one of SEQ ID NO: 212, 213, 215, 217, 220 or 524.

In some embodiments, the cell expressing the TCR further comprises a surrogate marker, such as a cell surface marker, which can be used to confirm transduction or engineering of the cell to express the TCR, such as a truncated cell surface receptor. In some aspects, the marker comprises all or a portion (e.g., a truncated form) of CD34, NGFR, Her2, or an epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence, such as T2A. See WO2014031687. In some embodiments, constructs encoding a TCR and a surrogate marker separated by a T2A, P2A or other ribosomal switch can express both proteins from the same construct, allowing the surrogate marker to be used as a marker to detect cells expressing such constructs. Examples of such markers that can be used are described below.

In some embodiments, the TCR or antigen-binding fragment thereof is encoded by a nucleotide sequence that is or has been codon-optimized and/or modified to eliminate hidden splice sites. Exemplary codon-optimized variants are described elsewhere herein.

B. Antibodies

In some embodiments, the binding molecule is an antibody or antigen-binding fragment thereof containing any one or more of the CDRs described above for TCR.

In some embodiments, the antibodies or antigen-binding fragments contain variable heavy and light chains containing CDR1, CDR2 and/or CDR3 contained in the alpha chain and CDR1, CDR2 and/or CDR3 contained in the beta chain, as shown in Table 3 or Table 5. The antibodies or antigen-binding fragments provided are also antibodies or antigen-binding fragments containing sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences.

In some embodiments, the antibody or antigen-binding fragment contains a variable region containing complementary determining region 1 (CDR-1), CDR-2 and CDR-3, which respectively contain the CDR-1, CDR-2 and CDR-3 amino acid sequences contained in the Vα region amino acid sequence shown in SEQ ID NO: 247. In some aspects, the antibody or antigen-binding fragment contains a variable region containing complementary determining region 1 (CDR-1), CDR-2 and CDR-3, which respectively contain the CDR-1, CDR-2 and CDR-3 amino acid sequences contained in the Vβ region amino acid sequence shown in SEQ ID NO: 258. The antibodies or antigen-binding fragments provided are also antibodies or antigen-binding fragments containing sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences.

In some embodiments, the antibodies or antibody fragments provided are human antibodies. In some embodiments, the antibodies or antibody fragments provided contain a _VH region containing a portion having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human heavy chain V segment, a portion having at least 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence encoded by a germline nucleotide human heavy chain D segment, and/or a portion having at least 95%, 96%, 97%, 98%, 99% or 100% identity to an amino acid sequence encoded by a germline nucleotide human heavy chain J segment; and/or a portion having a V _L region, which contains a portion having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence encoded by the germline nucleotide human κ or λ chain V segment and/or a portion having at least 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence encoded by the germline nucleotide human κ or λ chain J segment. In some embodiments, the portion of the _VH region corresponds to CDR-H1, CDR-H2 and/or CDR-H3. In some embodiments, the portion of the _VH region corresponds to framework region 1 (FR1), FR2, FR2 and/or FR4. In some embodiments, the portion of the _VL region corresponds to CDR-L1, CDR-L2 and/or CDR-L3. In some embodiments, the portion of the _VL region corresponds to FR1, FR2, FR2 and/or FR4.

In some embodiments, the antibody or antigen-binding fragment contains a framework region that contains a human germline gene segment sequence. For example, in some embodiments, the antibody or antigen-binding fragment contains a _VH region, wherein the framework regions, such as FR1, FR2, FR3 and FR4, have at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the framework regions encoded by human germline antibody segments, such as V and/or J segments. In some embodiments, the human antibody contains a _VL region, wherein the framework regions, such as FR1, FR2, FR3 and FR4, have at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the framework regions encoded by human germline antibody segments, such as V and/or J segments. For example, in some such embodiments, the framework sequence of the _VH and/or _VL sequences differs by no more than 10 amino acids, such as no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, compared to the framework region encoded by the human germline antibody segment. In some embodiments, the antibodies and antigen-binding fragments thereof, such as TCR-like antibodies, specifically recognize peptide antigenic determinants in the context of an MHC molecule, such as MHC class I. In some cases, the MHC class I molecule is an HLA-A2 molecule, such as HLA-A2*01.

In some embodiments, the antibody or antigen-binding fragment thereof recognizes or binds to an antigenic determinant or region of HPV16 E6, such as a peptide antigenic determinant containing an amino acid sequence shown in any one of SEQ ID NOs: 267-269. In some cases, the TCR or antigen-binding fragment thereof that recognizes or binds to a peptide antigenic determinant derived from HPV16 E6 is or comprises the sequence shown in SEQ ID NO: 268.

In some aspects, the TCR or antigen-binding fragment recognizes or binds to an antigenic determinant or region of the HPV16 E7 protein, such as a peptide antigenic determinant containing an amino acid sequence as shown in any one of SEQ ID NOs: 270-274. In some embodiments, the TCR or antigen-binding fragment thereof does not recognize or bind to an antigenic determinant E7 (11-19) comprising the amino acid sequence YMLDLQPET (SEQ ID NO: 271). In some cases, the peptide derived from HPV16 E7 is or contains the sequence shown in SEQ ID NO: 270.

Therefore, in some embodiments, anti-HPV antibodies are provided, including functional antibody fragments. In some embodiments, the antibody _VH and/or _VL domains, or their antigen binding sites, are capable of specifically binding to the peptide antigenic determinants of HPV 16. In some embodiments, the antibody comprises a variable heavy chain and a variable light chain, such as scFv. The antibody includes an antibody that specifically binds to HPV, such as HPV 16 E6 or HPV 16 E7. The anti-HPV antibodies provided are human antibodies. The antibodies include isolated antibodies. Molecules containing such antibodies are also provided, such as single chain proteins, fusion proteins and/or recombinant receptors, such as chimeric receptors, including antigen receptors.

As used herein, the term "antibody" is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragments: antigen-binding (Fab) fragments, F(ab') ₂ fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain ( _VH ) regions capable of specifically binding antigen, single-chain antibody fragments, including single-chain variable fragments (scFv) and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heterojunction antibodies, multispecific (e.g., bispecific) antibodies, bifunctional antibodies, trifunctional antibodies, and tetrafunctional antibodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

In some embodiments, the heavy chain and light chain of the antibody may be full length or may be an antigen binding portion (Fab, F(ab')2, Fv or single chain Fv fragment (scFv)). In other embodiments, the antibody heavy chain constant region is selected from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE, in particular, selected from, for example, IgG1, IgG2, IgG3 and IgG4, more particularly IgG1 (e.g., human IgG1). In another embodiment, the antibody light chain constant region is selected from, for example, κ or λ, in particular κ.

The antibodies provided are antibody fragments. "Antibody fragments" refer to non-intact antibody molecules that include a portion of an intact antibody that binds to an antigen bound by the intact antibody. Examples of antibody fragments include (but are not limited to) Fv, Fab, Fab', Fab'-SH, F(ab') ₂ ; bifunctional antibodies; linear antibodies; variable heavy chain ( _VH ) regions; single-chain antibody molecules, such as scFv and single-domain _VH monoclonal antibodies; and multispecific antibodies formed from antibody fragments. In specific embodiments, the antibody is a single-chain antibody fragment, such as scFv, that includes a heavy chain variable region and/or a light chain variable region.

The term "variable region" or "variable domain" when used in relation to an antibody, such as an antibody fragment, refers to a domain of the antibody heavy or light chain that is involved in the binding of the antibody to an antigen. The variable domains of the heavy and light chains ( _VH and _VL , respectively) of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single _VH or _VL domain is sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated by screening a library of complementary _VL or _VH domains, respectively, using _VH or _VL domains from antibodies that bind to that antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

A single domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody.

Antibody fragments can be made by a variety of techniques, including (but not limited to) proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, such as a fragment comprising a configuration that does not occur in nature, such as a fragment containing two or more antibody regions or chains connected by a synthetic linker, such as a peptide linker, and/or a fragment that is not produced by enzymatic digestion of a naturally occurring intact antibody. In some aspects, the antibody fragment is a scFv.

The anti-HPV antibodies provided are human antibodies. "Human antibodies" are antibodies having an amino acid sequence corresponding to an antibody produced by a human or human cell, or antibodies of non-human origin that utilize the human antibody repertoire or other human antibody coding sequences (including human antibody libraries). The term does not include humanized forms of non-human antibodies that contain non-human antigen binding regions, such as antibodies in which all or substantially all CDRs are non-human CDRs. The term includes antigen-binding fragments of human antibodies.

A "humanized" antibody is one in which all or substantially all of the CDR amino acid residues are derived from non-human CDRs and all or substantially all of the FR amino acid residues are derived from human FRs. A humanized antibody may optionally include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a non-human antibody variant that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some of the FR residues in the humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic attack. Such animals typically contain all or part of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci are generally inactive. Human antibodies can also be derived from human antibody libraries, including phage display libraries and cell-free libraries, which contain antibody coding sequences derived from human repertoires.

The antibodies provided are monoclonal antibodies, including monoclonal antibody fragments. As used herein, the term "monoclonal antibody" refers to an antibody obtained from or within a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for potential variants that occur during the generation of the monoclonal antibody preparation, which variants are usually present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different antigenic determinants, each monoclonal antibody of a monoclonal antibody preparation is directed against a single antigenic determinant on an antigen. The term should not be construed as requiring the production of antibodies by any particular method. Monoclonal antibodies can be produced by a variety of techniques, including (but not limited to) self-hybridoma production, recombinant DNA methods, phage display, and other antibody display methods.

As used herein, reference to a "corresponding form" of an antibody means that when comparing a property or activity of two antibodies, the same antibody form is used to compare the property. For example, if a statement is made that the activity of an antibody is greater than the activity of a corresponding form of a first antibody, it means that the activity of a particular form of the antibody (such as an scFv) is greater than the scFv form of the first antibody.

"Effector function" refers to the biological activity attributable to the Fc region of an antibody, which varies with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

In some embodiments, antibodies, such as antibody fragments, may contain at least a portion of an immunoglobulin constant region, such as one or more constant regions. In some embodiments, the constant region includes a light chain constant region and/or a heavy chain constant region 1 ( _CH1 ). In some embodiments, the antibody includes a _CH2 and/or _CH3 domain, such as an Fc region. In some embodiments, the Fc region is the Fc region of human IgG (such as IgG1 or IgG4).

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is based on the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

The terms "full-length antibody", "intact antibody" and "complete antibody" are used interchangeably herein to refer to an antibody whose structure is substantially similar to a native antibody structure or has a heavy chain containing an Fc region as defined herein.

An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

1. Mutants and Modifications

In certain embodiments, an antibody or antigen-binding fragment thereof comprises one or more amino acid changes, such as substitutions, deletions, insertions and/or mutations, compared to the sequence of an antibody described herein. Exemplary variants include variants designed to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be made to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

In certain embodiments, the antibody comprises one or more amino acid substitutions, for example, compared to the antibody sequences described herein and/or compared to sequences of a natural lineage, such as a human lineage. Sites of interest for substitutional mutagenesis include CDRs and FRs. Amino acid substitutions can be introduced into antibodies of interest and the products screened for desired activities, such as retained/improved antigen binding, reduced immunogenicity, improved half-life, and/or improved effector function, such as the ability to promote antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

In some embodiments, one or more residues within a CDR of a parent antibody (e.g., a humanized antibody or a human antibody) are substituted. In some embodiments, the substitution is made so as to restore the sequence or position in the sequence to a germline sequence, such as an antibody sequence found in the germline (e.g., the human germline), for example, to reduce the potential for immunogenicity, such as when administered to a human subject.

In some embodiments, changes are made in CDR "hotspot" residues encoded by codons that undergo high frequency mutation during the in vivo cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)) and/or residues that contact antigen, and the resulting variant _VH or _VL is tested for binding affinity. Affinity maturation by construction of secondary libraries and reselection from the secondary library has been described, e.g., in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide directed mutagenesis induction). A secondary library can then be created and screened to identify any antibody variants with the desired affinity. Another method of introducing diversity includes a CDR-directed approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis induction or modeling. CDR-H3 and CDR-L3 are often particularly targeted.

In certain embodiments, substitutions, insertions or deletions may occur within one or more CDRs, as long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such changes may be made, for example, outside of the residues in the CDRs that contact the antigen. In certain embodiments of the variant _VH and _VL sequences provided above, each CDR is unchanged or contains no more than one, two or three amino acid substitutions.

Amino acid sequence insertions include amino-terminal and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusions of the antibody N- or C-terminus to enzymes or polypeptides that increase the serum half-life of the antibody.

In certain embodiments, the antibody or antigen-binding fragment thereof is altered to increase or decrease the degree of glycosylation of the antibody, for example by changing the amino acid sequence to remove or insert one or more glycosylation sites and/or by modifying the oligosaccharides linked to the glycosylation sites, for example using certain cell lines.

Exemplary modifications, variants, and cell lines are described in, for example, patent publications No. US 2003/0157108; No. US 2004/0093621; No. US 2003/0157108; No. WO 2000/61739; No. WO 2001/29246; No. US 2003/0115614; No. US 2002/0164328; No. US 2004/0093621; No. US 2004/0132140; No. US 2004/0110704; No. US 2004/0110282; No. US 2004/0109865; No. WO 2003/085119; No. WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94 (4): 680-688 (2006); and WO2003/085107); WO 2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.); WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

A modified antibody is an antibody having one or more amino acid modifications in the Fc region, such as an antibody having a human Fc region sequence or other portion of a constant region (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

Such modifications may be made, for example, to improve half-life, alter binding to one or more types of Fc receptors, and/or alter effector function.

Variants are also cysteine engineered antibodies, such as "thioMAbs" and other cysteine engineered variants, in which one or more residues of the antibody are substituted with cysteine residues to generate reactive thiol groups at available sites, such as for binding agents and linker-agents to generate immunoconjugates. Cysteine engineered antibodies are described, for example, in U.S. Patent Nos. 7,855,275 and 7,521,541.

In some embodiments, the antibody is modified to contain other non-protein moieties, including water-soluble polymers. Exemplary polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, polydextrose, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and polydextrose or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polyoxypropylene/ethylene oxide copolymers, polyoxyethylene polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, the polymers can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

2. TCR-like CAR

In some embodiments, the antibody or its antigen-binding portion is expressed on a cell as a recombinant receptor, such as a portion of an antigen receptor. The antigen receptor is a functional non-TCR antigen receptor, such as a chimeric antigen receptor (CAR). Generally, a CAR containing an antibody or antigen-binding fragment that exhibits TCR-like specificity for a peptide in the context of an MHC molecule can also be referred to as a TCR-like CAR.

Thus, the binding molecules provided, such as HPV 16 E6 or E7 binding molecules, are antigen receptors, such as antigen receptors comprising one of the provided antibodies, such as TCR-like antibodies. In some embodiments, antigen receptors and other chimeric receptors specifically bind to regions or antigenic determinants of HPV16 E6 or E7, such as antigen receptors containing provided anti-HPV 16 E6 or E7 antibodies or antibody fragments, such as TCR-like antibodies. Antigen receptors are functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Cells expressing CARs and their use in transfer cell therapy are also provided, such as for the treatment of diseases and disorders associated with HPV 16 E6 or E7 expression.

Thus, provided herein are TCR-like CARs containing non-TCR molecules that exhibit T cell receptor specificity, such as for T cell antigenic determinants or peptide antigenic determinants in the context of MHC molecules when displayed or presented. In some embodiments, TCR-like CARs may contain antibodies or antigen binding portions thereof, such as TCR-like antibodies, as described herein. In some embodiments, antibodies or antibody binding portions thereof are reactive to specific peptide antigenic determinants in the context of MHC molecules, wherein the antibodies or antibody fragments can distinguish specific peptides in the context of MHC molecules from separate MHC molecules, separate specific peptides, and in some cases, unrelated peptides in the context of MHC molecules. In some embodiments, antibodies or antigen binding portions thereof may exhibit higher binding affinity than T cell receptors.

Exemplary antigen receptors, including CARs, and methods for engineering such receptors and introducing them into cells include those described in, for example, International Patent Application Publication Nos. WO2000/14257, WO2013/126726, WO2012/129514, WO2014/031687, WO2013/166321, WO2013/071154, WO2013/123061; U.S. Patent Application Publication Nos. US2002/131960, US2013/287748, US2013/0149337; U.S. Patent Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645 , 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118; and European Patent Application No. EP2537416; and/or Sadelain et al., Cancer Discov. 2013 Apr;3(4):388-398; Davila et al. (2013) PLoS ONE 8(4):e61338; Turtle et al., Curr. Opin. Immunol. , 2012 Oct;24(5):633-39; Wu et al., Cancer , March 18(2): 160-75, 2012. In some aspects, the antigen receptor includes a CAR as described in U.S. Patent No. 7,446,190, and an antigen receptor as described in International Patent Application Publication No. WO2014/055668 A1. Exemplary CARs include CARs disclosed in any of the aforementioned publications (e.g., WO2014/031687, US 8,339,645, US 7,446,179, US 2013/0149337, U.S. Patent No. 7,446,190, U.S. Patent No. 8,389,282), for example, wherein the antigen binding portion (e.g., scFv) is replaced by an antibody, such as provided herein.

In some embodiments, CARs generally include an extracellular antigen (or ligand) binding domain, such as an antibody or antigen binding fragment thereof specific for a peptide in the context of an MHC molecule, which is linked to one or more intracellular signaling components, in some embodiments via a linker and/or a transmembrane domain. In some embodiments, such molecules generally mimic or emulate the signaling through natural antigen receptors such as TCRs, and, as appropriate, through the combination of such receptors and co-stimulatory receptors.

In some embodiments, CARs typically include one or more antigen binding molecules, such as one or more antigen binding fragments, domains or portions, or one or more antibody variable domains and/or antibody molecules in their extracellular portion. In some embodiments, CARs include antigen binding portions or antibody molecule portions, such as single-chain antibody fragments (scFv) derived from variable heavy chains (VH) and variable light chains (VL) of monoclonal antibodies (mAbs). In some embodiments, CARs contain TCR-like antibodies, such as antibodies or antigen binding fragments (e.g., scFv) that specifically recognize peptide antigen determinants present on the cell surface in the context of MHC molecules.

In some embodiments, the antigen-specific binding or recognition component is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the CAR includes a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain that naturally associates with one of the domains in the CAR is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

In some embodiments, the transmembrane domain is derived from a natural or synthetic source. In the case of a natural source, in some aspects, the domain is derived from any membrane-bound protein or transmembrane protein. The transmembrane region includes a transmembrane region derived from (i.e., at least comprising a transmembrane region of) the alpha, beta or zeta chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain comprises primarily hydrophobic residues, such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain.

In some embodiments, a short oligo- or polypeptide linker is present, such as a linker between 2 and 10 amino acids in length, such as a linker containing glycine and serine, such as a glycine-serine dimer, and forms a connection between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

In some embodiments, CAR, such as TCR-like CAR, such as the antibody portion thereof, further includes a spacer, which may be or include at least a portion of an immunoglobulin constant region or a variant or modified version thereof, such as a hinge region, such as an IgG4 hinge region, and/or _CH1 / _CL and/or Fc region. In some embodiments, the constant region or portion belongs to human IgG, such as IgG4 or IgG1. In some aspects, the portion of the constant region serves as a spacer between an antigen recognition component (e.g., scFv) and a transmembrane domain. The length of the spacer can increase the reactivity of the cell after antigen binding compared to the absence of the spacer. In some examples, the spacer is or is about 12 amino acids long or is no more than 12 amino acids long. Exemplary spacers include spacers having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, including any integer between the endpoints of any of the listed ranges. In some embodiments, the spacer has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include an IgG4 hinge alone, an IgG4 hinge linked to the _CH2 and _CH3 domains, or an IgG4 hinge linked to the _CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res. , 19:3153 or International Patent Application Publication No. WO2014/031687.

In some embodiments, the constant region or part belongs to human IgG, such as IgG4 or IgG1. In some embodiments, the spacer has the sequence ESKYGPPCPPCP (shown in SEQ ID NO: 314) and is encoded by the sequence shown in SEQ ID NO: 295. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 296. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 297. In some embodiments, the constant region or part belongs to IgD. In some embodiments, the spacer has the sequence shown in SEQ ID NO: 298. In some embodiments, the spacer has an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to any of SEQ ID NOs: 296, 297, 298, or 314.

The antigen recognition domain is typically linked to one or more intracellular signaling components, such as signaling components that simulate activation via an antigen receptor complex (such as a TCR complex) and/or transmit signals via another cell surface receptor in the case of a CAR. Therefore, in some embodiments, the antibody or antigen binding fragment thereof is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that naturally binds to one of the domains in a receptor (such as a CAR) is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

In some embodiments, the transmembrane domain is derived from a natural or synthetic source. In the case of a natural source, in some aspects, the domain is derived from any membrane-bound protein or transmembrane protein. The transmembrane region includes a transmembrane region derived from (i.e., at least comprising its transmembrane region): α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain mainly comprises hydrophobic residues, such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain. In some embodiments, the connection is via a linker, a spacer and/or a transmembrane domain.

An intracellular signaling domain is an intracellular signaling domain that mimics or imitates signaling through natural antigen receptors, signaling through such receptors in combination with co-stimulatory receptors, and/or signaling through co-stimulatory receptors alone. In some embodiments, a short oligopeptide or polypeptide linker is present, such as a linker between 2 and 10 amino acids in length, such as a linker containing glycine and serine, such as a glycine-serine dimer, and the linker forms a connection between the transmembrane domain of the CAR and the cytoplasmic signaling domain.

CAR generally includes at least one intracellular signaling component or multiple components. In some embodiments, CAR includes an intracellular component of the TCR complex, such as a TCR CD3+ chain that mediates T cell activation and cytotoxicity, such as a CD3ζ chain. Therefore, in some embodiments, the antigen binding molecule is connected to one or more cell signaling modules. In some embodiments, the cell signaling module includes a CD3 transmembrane domain, a CD3 intracellular signaling domain, and/or other CD transmembrane domains. In some embodiments, CAR further includes a portion of one or more other molecules, such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some embodiments, CAR includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

In some embodiments, when attached to a CAR, the cytoplasmic domain or intracellular signaling domain of the CAR activates an immune cell, such as at least one of the normal effector functions or responses of a T cell engineered to express the CAR. For example, in some cases, the CAR induces T cell functions, such as cytolytic activity; or T helper cell activities, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an antigen receptor component or intracellular signaling domain of a co-stimulatory molecule is used in place of a complete immunostimulatory chain, such as when the portion transduces an effector function signal. In some embodiments, the intracellular signaling domain(s) include a cytoplasmic sequence of a T cell receptor (TCR), and in some embodiments, also include a cytoplasmic sequence of a co-receptor that cooperates with such receptors in the natural environment to initiate signal transduction after antigen receptor engagement, and/or any derivatives or variants of such molecules, and/or any synthetic sequence with the same functional capability.

In the case of natural TCR, full activation generally requires not only signaling via TCR, but also synergistic stimulation signals. Therefore, in some embodiments, in order to promote full activation, CAR also includes components for generating secondary or synergistic stimulation signals. In other embodiments, CAR does not include components for generating synergistic stimulation signals. In some aspects, another CAR is expressed in the same cell and provides components for generating secondary or synergistic stimulation signals. In some aspects, the cell comprises a first CAR and a second CAR, the first CAR contains a signaling domain to induce a primary signal, and the second CAR binds to a second antigen and contains components for generating synergistic stimulation signals. For example, the first CAR may be an activation CAR and the second CAR may be a synergistic stimulation CAR. In some embodiments, in order to induce a specific effector function in a cell, both CARs must be linked, which can provide specificity and selectivity for the targeted cell type.

T cell activation is described in some aspects as being mediated by two types of cytoplasmic signaling sequences: cytoplasmic signaling sequences that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and cytoplasmic signaling sequences that act in an antigen-independent manner to provide secondary or synergistic stimulatory signals (secondary cytoplasmic signaling sequences). In some aspects, a CAR includes one or both of these signaling components.

In some embodiments, the CAR includes an initial cytoplasmic signaling sequence that regulates the primary activation of the TCR complex. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif, which is called an immunoreceptor tyrosine-based activation motif or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs include primary cytoplasmic signaling sequences derived from: TCR or CD3ζ, FcRγ, CD3γ, CD3δ, or CD3ε. In some embodiments, the cytoplasmic signaling molecule in the CAR contains a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3ζ.

In some embodiments, the CAR includes a signaling domain and/or a transmembrane portion of a co-stimulatory receptor (such as CD28, 4-1BB, OX40, DAP10, and ICOS). In some embodiments, the same CAR includes both an activation component and a co-stimulatory component; in other embodiments, the activation domain is provided by one CAR, and the co-stimulatory component is provided by another CAR that recognizes another antigen.

In some embodiments, the activation domain is included in one CAR, and the synergistic stimulatory component is provided by another chimeric receptor that recognizes another antigen. In some embodiments, the CAR includes an activating or stimulating CAR and a synergistic stimulatory receptor, both of which are expressed on the same cell (see WO2014/055668). In some aspects, the receptor containing HPV 16 E6 or E7 antibodies is a stimulating or activating CAR; in other aspects, it is a synergistic stimulatory receptor. In some embodiments, the cell further comprises an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine , 5(215) (December 2013)), such as an inhibitory receptor that recognizes a peptide antigenic determinant other than HPV 16 E6 or HPV16 E7, wherein the activation signal delivered by the CAR targeting HPV16 is attenuated or inhibited by binding of the inhibitory CAR to its ligand, for example to reduce off-target effects.

In some embodiments, cells expressing provided TCRs or other binding molecules further express other receptors, such as receptors capable of delivering co-stimulatory or pro-survival signals, such as co-stimulatory receptors (see WO2014/055668); and/or blocking or altering the outcome of inhibitory signals, such as signals normally delivered via immune checkpoints or other immunosuppressive molecules (such as those expressed in the tumor microenvironment), for example, to promote the increased efficacy of such engineered cells. See, e.g., Tang et al., Am J Transl Res. 2015; 7(3): 460-473. In some embodiments, the cell may further include one or more other exogenous or recombinant or engineered components, such as one or more exogenous factors and/or co-stimulatory ligands, which are expressed on or in the cells or secreted by the cells and can promote function, such as in a microenvironment. Examples of such ligands and components include, for example, TNFR and/or Ig family receptors or ligands, such as 41BBL, CD40, CD40L, CD80, CD86, interleukins, chemokines and/or antibodies or other molecules, such as scFv. See, for example, patent application publications WO2008121420 A1, WO2014134165 A1, and US20140219975 A1. In some embodiments, the cells comprise one or more inhibitory receptors (iCAR, see Fedorov et al., Sci. Transl. Medicine , 5(215) (December 2013)), such as inhibitory receptors that bind to a ligand or antigen that is not associated with the disease or condition or is not expressed therein or thereon.

In some embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-ζ) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) synergistic stimulatory domain linked to a CD3ζ intracellular domain.

In some embodiments, the CAR includes one or more, such as two or more, synergistic stimulatory domains and activation domains, such as primary activation domains, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-ζ, CD28, and 4-1BB.

In some embodiments, cells expressing CAR or other antigen receptors further include a surrogate marker, such as a cell surface marker, which can be used to confirm transduction or engineering of cells to express receptors, such as truncated cell surface receptors. In some aspects, the marker includes all or a portion (e.g., a truncated form) of CD34, NGFR, Her2, or an epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence, such as T2A. See WO2014031687. In some embodiments, the introduction of constructs encoding a CAR and a surrogate marker separated by a T2A ribosomal switch can express both proteins from the same construct, such that the surrogate marker can be used as a marker to detect cells expressing such a construct. In some embodiments, the marker and optionally a linker sequence can be any of those disclosed in Published Patent Application No. WO2014031687. For example, the marker can be a truncated cell surface receptor optionally linked to a linker sequence, such as a T2A cleavable linker sequence. Exemplary polypeptides of truncated EGFR (e.g., tEGFR) include the amino acid sequence set forth in SEQ ID NO: 299 or 300, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 299 or 300. Exemplary T2A linker sequences include the amino acid sequence set forth in SEQ ID NO: 302 or 301, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 302 or 301.

In some embodiments, the marker is a molecule that does not naturally occur on a T cell or does not naturally occur on the surface of a T cell, such as a cell surface protein, or a portion thereof.

In some embodiments, the molecule is a non-self molecule, such as a non-self protein, i.e., a molecule that cannot be recognized as "self" by the immune system of the host to which the cell is transferred.

In some embodiments, the marker has no therapeutic function and/or does not produce an effect other than being used as a marker for genetic engineering (e.g., for selecting successfully engineered cells). In other embodiments, the marker may be a therapeutic molecule or a molecule that otherwise exerts some desired effect, such as a ligand encountered by cells in vivo, such as a co-stimulatory or immune checkpoint molecule to enhance and/or weaken the cell response during donor-receptor transfer and encounter with a ligand.

In some cases, CARs are referred to as first, second, and/or third generation CARs. In some embodiments, first generation CARs are CARs that provide only CD3 chain-induced signals upon antigen binding; in some embodiments, second generation CARs are CARs that provide such signals and co-stimulatory signals, such as CARs that include intracellular signaling domains from co-stimulatory receptors (such as CD28 or CD137); in some embodiments, third generation CARs are CARs that include multiple co-stimulatory domains of different co-stimulatory receptors.

In some embodiments, the chimeric antigen receptor comprises an extracellular portion comprising a TCR-like antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv and the intracellular domain comprises an ITAM. In some aspects, the intracellular signaling domain comprises the signaling domain of the ζ chain of the CD3-zeta (CD3ζ) chain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain connecting the extracellular domain and the intracellular signaling domain. In some aspects, the transmembrane domain contains the transmembrane portion of CD28. The extracellular domain and the transmembrane domain can be directly or indirectly connected. In some embodiments, the extracellular domain and the transmembrane domain are connected by a spacer, such as any spacer described herein. In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell co-stimulatory molecule, such as an intracellular domain between a transmembrane domain and an intracellular signaling domain. In some embodiments, the T cell co-stimulatory molecule is CD28 or 41BB.

For example, in some embodiments, the CAR contains a TCR-like antibody as provided herein, such as an antibody fragment; a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof; and an intracellular signaling domain that contains a signaling portion of CD28 or a functional variant thereof and a signaling portion of CD3ζ or a functional variant thereof. In some embodiments, the CAR contains a TCR-like antibody as provided herein, such as an antibody fragment; a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof; and an intracellular signaling domain that contains a signaling portion of 4-1BB or a functional variant thereof and a signaling portion of CD3ζ or a functional variant thereof. In some such embodiments, the CAR further comprises a spacer comprising an Ig molecule, such as a portion of a human Ig molecule, such as an Ig hinge, for example, an IgG4 hinge, such as a spacer comprising only a hinge.

In some embodiments, the transmembrane domain of the receptor, such as a TCR-like CAR, is a transmembrane domain of human CD28 (e.g., accession number P0 1747.1) or a variant thereof, such as a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 303 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with SEQ ID NO: 303. In some embodiments, the transmembrane domain containing the CAR portion comprises the amino acid sequence shown in SEQ ID NO: 304 or an amino acid sequence that has at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with SEQ ID NO: 304.

In some embodiments, the intracellular signaling component of a CAR, such as a TCR-like CAR, contains an intracellular co-stimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as a domain having a LL to GG substitution at positions 186-187 of a native CD28 protein. For example, the intracellular signaling domain may comprise an amino acid sequence as set forth in SEQ ID NO: 305 or 306, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 305 or 306. In some embodiments, the intracellular domain comprises the intracellular synergistic stimulatory signaling domain of 4-1BB (e.g., accession number Q07011.1) or a functional variant or portion thereof, such as the amino acid sequence shown in SEQ ID NO: 307 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with SEQ ID NO: 307.

In some embodiments, the intracellular signaling domain of a CAR, such as a TCR-like CAR, comprises a human CD3ζ stimulatory signaling domain or a functional variant thereof, such as the 112 AA cytoplasmic domain of human CD3ζ isoform 3 (Deposit No.: P20963.2) or a CD3ζ signaling domain, as described in U.S. Patent No. 7,446,190 or U.S. Patent No. 8,911,993. For example, in some embodiments, the intracellular signaling domain comprises an amino acid sequence of SEQ ID NO: 308, 309, or 310, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to SEQ ID NO: 308, 309, or 310.

In some aspects, the spacer contains only the hinge region of IgG, such as only the hinge of IgG4 or IgG1, such as the spacer containing only the hinge as shown in SEQ ID NO: 314. In other embodiments, the spacer is or contains an Ig hinge, such as an IgG4-derived hinge, optionally linked to the CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, such as an IgG4 hinge, which is linked to the CH2 and CH3 domains, such as shown in SEQ ID NO: 297. In some embodiments, the spacer is an Ig hinge, such as an IgG4 hinge, which is linked to the CH3 domain, such as shown in SEQ ID NO: 296. In some embodiments, the spacer is or comprises a glycine-serine-rich sequence or other flexible linker, such as a known flexible linker.

For example, in some embodiments, TCR-like CARs include TCR-like antibodies or fragments, such as any TCR-like antibodies or fragments provided herein, including scFv; spacers, such as any of the spacers containing Ig-hinge; CD28 transmembrane domain; CD28 intracellular signaling domain; and CD3ζ signaling domain. In some embodiments, TCR-like CARs include TCR-like antibodies or fragments, such as any TCR-like antibodies or fragments provided herein, including scFv; spacers, such as any of the spacers containing Ig-hinge; CD28 transmembrane domain; CD28 intracellular signaling domain; and CD3ζ signaling domain. In some embodiments, such TCR-like CAR constructs further include T2A ribosomal skipping elements and/or tEGFR sequences, such as downstream of CAR.

In some embodiments, such CAR constructs further include a T2A ribosomal skipping element and/or a tEGFR sequence, such as shown in SEQ ID NO: 301 or 302, and a tEGFR sequence shown in SEQ ID NO: 299 or 300, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with SEQ ID NO: 299, 300, 301 or 302.

In some embodiments, the CAR comprises an HPV 16 E6 or E7 antibody or fragment, such as any of the HPV 16 E6 or E7 antibodies, including sdAbs (e.g., containing only the _VH region) and scFvs described herein; a spacer, such as any of the spacers containing an Ig-hinge; a CD28 transmembrane domain; a CD28 intracellular signaling domain; and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an HPV 16 antibody or fragment, such as any of the HPV 16 E6 or E7 antibodies, including sdAbs and scFvs described herein; a spacer, such as any of the spacers containing an Ig-hinge; a CD28 transmembrane domain; a CD28 intracellular signaling domain; and a CD3 zeta signaling domain. In some embodiments, such CAR constructs further include a T2A ribosomal skipping element and/or a tEGFR sequence, for example, downstream of CAR.

II. Nucleic Acids, Vectors and Expression Methods

Also provided are nucleic acids encoding any of the provided binding molecules, such as TCRs or antigen binding fragments thereof or antibodies or antigen binding fragments thereof or CARs containing such antibodies, such as those described herein. Nucleic acids may include nucleic acids comprising naturally and/or non-naturally occurring nucleotides and bases, such as nucleic acids with backbone modifications. The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably and refer to nucleotide polymers. Such nucleotide polymers may contain natural and/or non-natural nucleotides and include (but are not limited to) DNA, RNA and PNA. "Nucleic acid sequence" refers to the linear sequence of nucleotides that make up a nucleic acid molecule or polynucleotide.

In some embodiments, the binding molecule (e.g., TCR) or its antigen binding portion can be a recombinantly produced natural protein or a mutant form thereof in which one or more properties, such as binding characteristics, have been altered. In some aspects, the nucleic acid is synthetic. In some cases, the nucleic acid is or contains cDNA. In some aspects, the nucleic acid molecule can be modified for use in the constructs described herein, such as for codon optimization. In some cases, the sequence can be designed to contain terminal restriction site sequences for the purpose of cloning into a vector.

In some embodiments, nucleic acid molecules encoding binding molecules (e.g., TCRs) can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of encoding nucleic acids within or isolated from one or more given cells. In some embodiments, TCRs are obtained from a biological source, such as from a cell, such as from a T cell (e.g., a cytotoxic T cell), a T cell fusion tumor, or other publicly available source. In some embodiments, TCRs can be derived from one of a variety of animal species, such as humans, mice, rats, or other mammals, such as typically humans. In some embodiments, T cells can be obtained from isolated cells in vivo, such as from normal (or healthy) individuals or diseased individuals, including T cells present in peripheral blood mononuclear cells (PBMC) or tumor infiltrating lymphocytes (TIL). In some embodiments, T cells can be cultured T cell fusion tumors or pure lines. For example, in some embodiments, in order to generate a vector encoding a TCR, the α chain and the β chain can be PCR amplified from the total cDNA isolated from the T cell pure line expressing the TCR of interest and cloned into the expression vector. In some embodiments, the α chain and the β chain can be produced synthetically. In some embodiments, the α chain and the β chain are cloned into the same vector.

In some embodiments, a TCR or its antigen-binding portion can be produced synthetically based on knowledge of the TCR sequence.

In some embodiments, the nucleic acid molecule contains a nucleic acid sequence encoding an α chain and/or a nucleotide sequence encoding a β chain.

In some embodiments, the nucleic acid sequence encoding the alpha chain comprises one of the following: residues 103-870 of SEQ ID NO: 1; residues 103-858 of SEQ ID NO: 2; residues 55-810 of SEQ ID NO: 39; residues 55-789 of SEQ ID NO: 40; residues 67-825 of SEQ ID NO: 71; residues 67-813 of SEQ ID NO: 72; residues 73-831 of SEQ ID NO: 97; residues 73-819 of SEQ ID NO: 98; residues 67-837 of SEQ ID NO: 120; residues 67-825 of SEQ ID NO: 121; residues 67-828 of SEQ ID NO: 143; residues 67-816 of SEQ ID NO: 144; NO: 163 residues 61-819; SEQ ID NO: 164 residues 61-807; SEQ ID NO: 189 residues 61-816; SEQ ID NO: 190 residues 67-840; or SEQ ID NO: 216 residues 64-828; SEQ ID NO: 241 residues 64-816; SEQ ID NO: 242 residues; or degenerate sequences thereof or sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity therewith. In some aspects, the nucleotide sequence encoding the beta strand comprises one of the following: residues 73-957 of SEQ ID NO: 16; residues 73-957 of SEQ ID NO: 17; residues 43-924 of SEQ ID NO: 52; residues 43-912 of SEQ ID NO: 53; residues 58-930 of SEQ ID NO: 82; residues 58-918 of SEQ ID NO: 83; residues 58-930 of SEQ ID NO: 108; residues 58-918 of SEQ ID NO: 109; residues 58-930 of SEQ ID NO: 131; residues 58-918 of SEQ ID NO: 132; residues 58-930 of SEQ ID NO: 151; residues 58-918 of SEQ ID NO: 152; NO: 174 residues 58-942; SEQ ID NO: 175 residues 58-930; SEQ ID NO: 200 residues 58-939; SEQ ID NO: 201 residues 58-927; SEQ ID NO: 226 residues 58-933; SEQ ID NO: 227 residues 58-921; SEQ ID NO: 252 residues 58-936; SEQ ID NO: 253 residues 58-924; degenerate sequences thereof or sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity therewith.

In some embodiments, the nucleotide sequence encoding the alpha chain and/or the nucleotide sequence encoding the beta chain is codon optimized. Typically, codon optimization involves balancing the percentage of codons selected from a large number of publicly available human transfer RNAs so that none is overloaded or restrictive. In some cases, this is necessary because most amino acids are encoded by more than one codon, and the usage of codons varies from organism to organism. Differences in codon usage between transfected genes and host cells have an impact on protein expression and immunogenicity of nucleic acid constructs. In general, for codon optimization, codons are selected to select those codons that are balanced with human usage frequency. Typically, the codon redundancy of amino acids causes different codons to encode one amino acid. In some embodiments, when a codon is selected for replacement, the resulting mutation is expected to be a silent mutation, so that the codon change does not affect the amino acid sequence. Generally, the last nucleotide of the codon can remain unchanged without affecting the amino acid sequence. In some cases, the nucleic acid sequence encoding the binding molecule, such as TCR or its antigen binding fragment, is modified so that the hidden splice site is removed. In some embodiments, a polynucleotide encoding the TCR shown in SEQ ID NO: 365 is provided herein.

In some cases, the nucleic acid sequence encoding the alpha chain contains one of the following: residues 103-907 of SEQ ID NO:3, residues 103-895 of SEQ ID NO:4, residues 55-810 of SEQ ID NO:41, residues 55-789 of SEQ ID NO:42, a degenerate sequence thereof, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity thereto. In some examples, the nucleotide sequence encoding the beta chain contains one of the following: residues 82-966 of SEQ ID NO: 18, residues 82-954 of SEQ ID NO: 19, residues 43-924 of SEQ ID NO: 54, residues 43-912 of SEQ ID NO: 55, degenerate sequences thereof, or sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity therewith.

In some embodiments, the nucleic acid molecule encoding the alpha chain and/or beta chain of the TCR comprises a nucleic acid sequence corresponding to SEQ ID NO: shown in Table 7. The nucleic acid molecule encoding the TCR provided is also a nucleic acid molecule containing a sequence that is at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such a sequence. Also provided is any one of the TCR alpha chains encoded by any one of the sequences shown in Table 7 , such as in each column thereof. Also provided is any one of the TCR beta chains encoded by any one of the sequences shown in Table 7, such as in each column thereof. Also provided is any one of the TCR alpha chains and beta chains encoded by any one of the sequences shown in Table 7 , such as in each column thereof. Exemplary TCRs or modified versions thereof encoded by such sequences are also shown in Table 7 .

In some embodiments, provided herein are nucleic acids encoding the TCR alpha chain set forth in SEQ ID NO:39 and/or the TCR beta chain set forth in SEQ ID NO:52. In some embodiments, provided herein are nucleic acids encoding the TCR alpha chain set forth in SEQ ID NO:39 and/or the TCR beta chain set forth in SEQ ID NO:361. In some embodiments, provided herein are polynucleotides encoding the TCR set forth in SEQ ID NO:69. In some embodiments, provided herein are polynucleotides encoding the TCR set forth in SEQ ID NO:363. In some embodiments, provided herein are polynucleotides encoding the TCR set forth in SEQ ID NO:389. In some embodiments, provided herein are polynucleotides encoding the TCR set forth in SEQ ID NO:365. In some embodiments, provided herein are polynucleotides encoding the TCR set forth in SEQ ID NO:402. The nucleic acids or polynucleotides provided herein are also nucleic acids or polynucleotides containing sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences.

In some embodiments, nucleic acids encoding TCR alpha chains shown in SEQ ID NO: 1, 39, 71, 97, 120, 143, 163, 189, 215 or 241 and/or nucleic acids encoding TCR beta chains shown in SEQ ID NO: 411, 361, 414, 415, 416, 417, 418, 419, 423 or 424 are provided herein. The nucleic acids provided herein or the polynucleotides provided herein are also nucleic acids or polynucleotides containing sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to such sequences. The embodiments provided are also one or more chains (e.g., alpha chains and/or beta chains) of a TCR or its binding fragment encoded by any of such polynucleotides.

Vectors or constructs containing such nucleic acid molecules are also provided. In some embodiments, the vector or construct contains one or more promoters operably linked to nucleotides encoding the alpha chain and/or the beta chain. In some embodiments, the promoter is operably linked to one or more nucleic acid molecules.

In some embodiments, a vector or construct may contain a single promoter that drives the expression of one or more nucleic acid molecules. In some embodiments, such promoters may be polycistronic (bicistronic or tricistronic, see, e.g., U.S. Patent No. 6,060,273). For example, in some embodiments, a transcription unit may be engineered as a bicistronic unit containing an internal ribosome entry site (IRES), which allows for co-expression of gene products (e.g., encoding the alpha and/or beta chains of a TCR) by the message from a single promoter. Alternatively, in some cases, a single promoter can direct the expression of RNA that contains two or three genes (e.g., encoding the alpha and/or beta chains of a TCR) in a single open reading frame (ORF) that are separated from each other by sequences encoding a self-cleaving peptide (e.g., T2A) or a protease recognition site (e.g., furin). Thus, the ORF encodes a single polyprotein that is cleaved into individual proteins during translation (in the case of 2A, such as T2A) or after translation. In some cases, peptides such as T2A can cause the ribosome to skip (ribosome skipping) the synthesis of the peptide bond at the C-terminus of the 2A element, resulting in a separation between the end of the 2A sequence and the next downstream peptide (see, e.g., de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and deFelipe et al. Traffic 5:616-626 (2004)). Examples of 2A cleavage peptides, including 2A cleavage peptides that can induce ribosome skipping, are Thosea asigna virus (T2A, e.g., SEQ ID NO: 301 or 302), porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 32 or 311), equine rhinitis virus type A (E2A, e.g., SEQ ID NO: 313), and the 2A sequence from foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 312), as described in U.S. Patent Publication No. 2007/0116690.

In some cases, the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a nucleotide sequence encoding an internal ribosome entry site (IRES) or a peptide sequence that causes ribosome skipping. In some cases, the nucleotide sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are separated by a peptide sequence that causes ribosome skipping. In some such cases, the peptide that causes ribosome skipping is a P2A or T2A peptide and/or contains the amino acid sequence shown in SEQ ID NO: 32, 301, 302 or 311. In some aspects, the nucleotide sequence encoding the peptide that causes ribosome skipping contains the sequence shown in SEQ ID NO: 31 or 315-326.

In some embodiments, the nucleic acid sequence encoding the alpha chain and the nucleotide sequence encoding the beta chain are present in any order, separated by a nucleotide sequence encoding an internal ribosome entry site (IRES) or a peptide sequence that causes ribosome skipping. For example, in some embodiments, the nucleic acid molecule comprises in the following order: a nucleic acid sequence encoding the beta chain; a nucleic acid sequence encoding an IRES or a peptide sequence that causes ribosome skipping, such as a P2A or T2A sequence as described herein; and a nucleic acid sequence encoding the alpha chain. In other embodiments, the nucleic acid molecule contains in the following order: a nucleic acid sequence encoding the alpha chain, a nucleic acid sequence encoding an IRES or a peptide sequence that causes ribosome skipping, and a nucleic acid sequence encoding the beta chain.

Thus, in some embodiments, the nucleic acid molecule encodes a polypeptide comprising a β-chain, an IRES or a peptide that causes ribosome skipping, and an α-chain in the following order. In other embodiments, the nucleic acid molecule encodes a polypeptide comprising an α-chain, an IRES or a peptide that causes ribosome skipping, and a β-chain in the following order.

In some embodiments, the nucleic acid molecule encodes a polypeptide containing an amino acid sequence shown in Table 8 , or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule encodes a polypeptide shown in any one of SEQ ID NOs: 37, 38, 69, 70, 95, 96, 118, 119, 141, 142, 161, 162, 187, 188, 213, 214, 239, 240, 265, or 266, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 33-36, 65-68, 93, 94, 116, 117, 139, 140, 159, 160, 185, 186, 211, 212, 237, 238, 263, 264, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, provided herein is a polynucleotide encoding a TCR as set forth in any one of SEQ ID NOs: 400, 363, 403, 404, 405, 406, 407, 408, 409, or 410. In some embodiments, provided herein are polynucleotides encoding the TCR set forth in any one of SEQ ID NOs: 364, 65, 93, 116, 139, 159, 185, 211, 237, or 263. In some embodiments, provided herein are polynucleotides encoding the TCR set forth in any one of SEQ ID NOs: 387, 389, 391, 392, 393, 394, 395, 396, 397, or 398. In some embodiments, provided herein are polynucleotides encoding the TCR set forth in SEQ ID NO: 399. In some embodiments, provided herein are polynucleotides encoding the TCR set forth in SEQ ID NO: 67. In some embodiments, provided herein are polynucleotides encoding the TCR set forth in SEQ ID NO: 388. In some embodiments, provided herein are polynucleotides encoding the TCR set forth in SEQ ID NO: 390. In some embodiments, provided herein is a polynucleotide encoding the TCR set forth in SEQ ID NO: 401. In some embodiments, provided herein is a polynucleotide encoding the TCR set forth in SEQ ID NO: 402. Also provided herein are nucleic acids or provided polynucleotides that contain sequences that are at least or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to such sequences. Also provided are embodiments of one or more chains (e.g., alpha chain and/or beta chain) of a TCR or its binding fragment encoded by any of such polynucleotides.

Also provided are polypeptides containing a sequence encoded by any of the provided nucleic acids. In some aspects, the polypeptide comprises an amino acid sequence corresponding to the SEQ ID NO. shown in Table 8 , or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the polypeptide comprises a sequence shown in any of SEQ ID NOs: 37, 38, 69, 70, 95, 96, 118, 119, 141, 142, 161, 162, 187, 188, 213, 214, 239, 240, 265 or 266, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the polypeptide comprises the sequence shown in SEQ ID NO: 362, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Exemplary such TCRs or modified versions thereof are also shown in Table 8 .

In some embodiments, the nucleic acid molecule can further encode a marker (e.g., EGFRt or other markers as described), which is separated from the CAR or from the TCR chain by a linker, such as a cleavable linker sequence or a peptide sequence that causes ribosome skipping, such as T2A or P2A.

In some embodiments, the constructs can be arranged in any order such that the sequence encoding the marker is 3' to the α and/or β sequence, 5' to the α and/or β sequence, and/or between the α and β sequences, wherein, in some cases, each individual component is separated by a cleavable linker sequence or a peptide that causes ribosome skipping (e.g., T2A or P2A) or an IRES. In some embodiments, the nucleic acid molecule contains a nucleic acid sequence encoding a marker (e.g., EGFRt), a cleavable linker or ribosome skipping sequence (e.g., T2A or P2A), a β strand, a cleavable linker or ribosome skipping sequence (e.g., T2A or P2A), and an α strand in the following order. In some embodiments, the nucleic acid molecule contains a nucleic acid sequence encoding a marker (e.g., EGFRt), a cleavable linker or ribosomal skipping sequence (e.g., T2A or P2A), an alpha chain, a cleavable linker or ribosomal skipping sequence (e.g., T2A or P2A), and a beta chain in the following order. In some embodiments, the nucleic acid molecule contains a nucleic acid sequence encoding a beta chain, a cleavable linker or ribosomal skipping sequence (e.g., T2A or P2A), an alpha chain, a cleavable linker or ribosomal skipping sequence (e.g., T2A or P2A), and a marker (e.g., EGFRt) in the following order. In some embodiments, the nucleic acid molecule contains a nucleic acid sequence encoding an alpha chain, a cleavable linker or a ribosomal skipping sequence (e.g., T2A or P2A), a beta chain, a cleavable linker or a ribosomal skipping sequence (e.g., T2A or P2A), and a marker (e.g., EGFRt) in the following order. In some embodiments, the nucleic acid molecule contains a nucleic acid sequence encoding an alpha chain, a cleavable linker or a ribosomal skipping sequence (e.g., T2A or P2A), a marker (e.g., EGFRt), a cleavable linker or a ribosomal skipping sequence (e.g., T2A or P2A), and a beta chain in the following order. In some embodiments, the nucleic acid molecule contains a nucleic acid sequence encoding a β chain, a cleavable linker or a ribosomal skipping sequence (e.g., T2A or P2A), a marker (e.g., EGFRt), a cleavable linker or a ribosomal skipping sequence (e.g., T2A or P2A), and an α chain in the following order.

In some embodiments, introduction of constructs encoding CAR and EGFRt separated by a T2A ribosomal switch allows expression of both proteins from the same construct, allowing EGFRt to be used as a marker for detecting cells expressing such constructs.

The nucleic acid may encode an amino acid sequence comprising a variable alpha (Vα) region or a variable light chain ( _VL ) region of a TCR or antibody, respectively. In some cases, the nucleic acid encodes an amino acid sequence comprising a variable beta (Vβ) region or a variable heavy chain ( _VH ) region of a TCR or antibody, respectively. In another embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided.

Vectors are also provided, such as vectors containing any of the nucleic acids described herein. In some embodiments, one or more nucleic acids encoding one or both chains of a binding molecule (e.g., a TCR) are cloned into one or more suitable expression vectors. The expression vector can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include vectors designed for propagation and expansion or for expression or for both, such as plasmids and viruses. In some embodiments, the vector is an expression vector.

In some embodiments, the vector may be a vector of the pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJolla, Calif.), pET series (Novagen, Madison, Wis.), pGEX series (Pharmacia Biotech, Uppsala, Sweden), or pEX series (Clontech, Palo Alto, Calif.). In some cases, phage vectors such as λG10, λGT11, λZapII (Stratagene), λEMBL4, and λNM1149 may also be used. In some embodiments, plant expression vectors may be used and include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some cases, the vector is a viral vector. In some such aspects, the viral vector is a retroviral vector, such as a lentiviral vector. In some cases, the lentiviral vector is derived from HIV-1.

In some embodiments, the recombinant expression vector can be prepared using standard recombinant DNA techniques. In some embodiments, the vector can contain regulatory sequences, such as transcriptional and translational start and stop codons, as appropriate and taking into account whether the vector is DNA-based or RNA-based, which are specific to the type of host (e.g., bacteria, fungi, plants, or animals) into which the vector is to be introduced. In some embodiments, the vector can contain a non-natural promoter operably linked to a nucleotide sequence encoding a binding molecule, such as a TCR, an antibody, or an antigen-binding fragment thereof. In some embodiments, the promoter may be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter present in the long terminal repeat sequence of the mouse stem cell virus. Other promoters known to those skilled in the art are also included.

Methods for making binding molecules (including antigen binding fragments) are also provided. In some embodiments, host cells containing such nucleic acids are provided. In order to recombinantly produce binding molecules, nucleic acids encoding binding molecules such as those described above can be isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be easily isolated and sequenced using known procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the alpha and beta chains of a TCR or the heavy and light chains of an antibody). In some embodiments, a method for producing a binding molecule is provided, wherein the method comprises: culturing a host cell comprising a nucleic acid encoding a binding molecule as provided above under conditions suitable for expressing the binding molecule; and recovering the binding molecule from the host cell (or host cell culture medium) as appropriate.

In one such embodiment, the host cell comprises a vector (e.g., the vector has been transformed) comprising a nucleic acid encoding an amino acid sequence comprising a Vβ region of a TCR or an antigen-binding fragment thereof and a nucleic acid encoding an amino acid sequence comprising a Vα region of a TCR or an antigen-binding fragment thereof. In another such embodiment, the host cell comprises a vector (e.g., the vector has been transformed) comprising a nucleic acid encoding an amino acid sequence comprising a VH of an antibody or an antigen-binding fragment thereof and a VL of an antibody or an antigen-binding fragment thereof. In some aspects, the host cell comprises (e.g., the vector has been transformed): a first vector comprising a nucleic acid encoding an amino acid sequence comprising a Vα region of a TCR or an antigen-binding fragment thereof; and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a Vβ region of a TCR or an antigen-binding fragment thereof. In some embodiments, the host cell comprises (e.g., has been transformed with): a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody or an antigen-binding fragment thereof; and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of an antibody or an antigen-binding fragment thereof.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable hosts for the selection or expression of molecular encoding vectors, including fungal and yeast strains whose glycosylation pathways have been modified to mimic or imitate the glycosylation pathways in human cells. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004); and Li et al., Nat. Biotech. 24: 210-215 (2006).

Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44.Lec13 CHO cells, and FUT8 CHO cells; ^PER.C6® cells; and NSO cells. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make the desired post-translational modification to the binding molecule. For example, in some embodiments, CHO cells produce polypeptides that are more sialylated than the same polypeptides produced in 293 cells. In some embodiments, the binding molecules are produced in a cell-free system. Exemplary cell-free systems are described in, e.g., Sitaraman et al., Methods Mol. Biol. 498:229-44 (2009); Spirin, Trends Biotechnol. 22:538-45 (2004); Endo et al., Biotechnol. Adv. 21:695-713 (2003).

1. Exemplary characteristics of binding molecules and engineered cells

In some aspects, the binding molecules provided, such as TCRs or TCR-like CARs, have one or more specified functional characteristics, such as binding properties, including binding to a specific antigenic determinant, no off-target binding or activity and/or a specific binding affinity. In some embodiments, any one or more of the characteristics of the provided TCRs can be assessed by expressing the TCR, for example, by introducing one or more nucleic acids encoding the TCR into T cells, such primary T cells, or T cell lines. In some embodiments, the T cell line is a Jurkat cell or a Jurkat-derived cell line. An exemplary Jurkat-derived cell line is the J.RT3-T3.5 (ATCC® TIB-153 ^™ ) cell line, which was generated by treating the Jurkat leukemia cell line with irradiation mutation induction and negatively selecting with the OKT3 monoclonal antibody (see Weiss and Stobo, J. Ex. Med. 160(5):1284-1299 (1984)).

In some embodiments, the binding molecules provided are capable of binding to a peptide epitope of HPV16 as described above, such as an epitope of HPV 16 E6 or E7, with at least some affinity, as measured by any of a variety of known methods. In some embodiments, the peptide epitope is a peptide in the context of an MHC molecule or ligand. In some embodiments, affinity is represented by an equilibrium dissociation constant ( _KD ) or an association constant ( _ka ). In some embodiments, affinity is represented by _EC50 .

In some embodiments, a binding molecule, e.g., a TCR, binds to a peptide epitope, e.g., in complex with an MHC molecule, e.g., specifically, with an affinity or _KA (i.e., the equilibrium association constant for a specific binding interaction, in units of 1/M; equal to the ratio of the association rate [ _kon or _ka ] to the dissociation rate [ _koff or _kd ] of the association reaction, assuming a bimolecular interaction) equal to or greater than 10 ⁵ M ^-1 . In some embodiments, a TCR or fragment thereof exhibits a binding affinity for a peptide ^epitope with a _KD (i.e., the equilibrium dissociation constant for a specific binding interaction, in units of M; equal to the ratio of the dissociation rate [ _koff or _kd ] to the association rate [ _kon or _ka ] of the association reaction, assuming a bimolecular interaction) equal to or less than 10 -5 M. For example, the equilibrium dissociation constant _KD is in the range of from or about ^10-5 M to or about ^10-12 M, such as from or about ^10-6 M to or about ^10-10 M, from or about ^10-7 M to or about ^10-11 M, from or about ^10-6 M to or about ^10-8 M, or from or about ^10-7 M to or about ^10-8 M. Association rates (association rate constant; _kon or _ka ; in units of 1/Ms) and dissociation rates (dissociation rate constant; _koff or _kd ; in units of 1/s) can be determined using any of the analytical methods known in the art, such as surface plasmon resonance (SPR).

In some embodiments, binding affinity can be classified as high affinity or low affinity. In some cases, binding molecules (e.g., TCRs) that exhibit low to medium affinity binding exhibit a _KA of at most 10 ⁷ M ^-1 , at most 10 ⁶ M ^-1 , at most 10 ⁵ M ^-1 . In some cases, binding molecules (e.g., TCRs) that exhibit high affinity binding to a particular antigenic determinant interact with such antigenic determinant with a KA of at least 10 ⁷ M ^-1 , at least 10 ⁸ M ^-1 , at least 10 ⁹ M ^-1 , at least 10 ¹⁰ M- ¹ , at _least 10 ¹¹ M ^-1 , at least 10 ¹² M ^-1 , or at least 10 ¹³ M ^-1 . In some embodiments, the binding affinity ( _EC50 ) and/or dissociation constant of the binding molecule to a peptide epitope of HPV 16 E6 or E7 is from or about 0.1 nM to 1 μM, 1 nM to 1 μM, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 10 nM, 10 nM to 500 nM, 10 nM to 100 nM, 10 nM to 50 nM, 50 nM to 500 nM, 50 nM to 100 nM, or 100 nM to 500 nM. In certain embodiments, the binding affinity ( _EC50 ) and/or dissociation constant of the binding molecule to a peptide epitope of HPV 16 E6 or E7 is or is about or is less than or is about 1 μM, 500 nm, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM.

A variety of assays are known for assessing binding affinity and/or determining whether a binding molecule specifically binds to a particular ligand (e.g., a peptide in the context of an MHC molecule). It is within the capabilities of one skilled in the art to determine the binding affinity of a binding molecule, such as a TCR, for a T cell epitope of a target polypeptide, such as by using any of a variety of binding assays well known in the art. For example, in some embodiments, a BIAcore machine may be used to determine the binding constant of a complex between two proteins. The dissociation constant of the complex may be determined by monitoring the change in refractive index versus time as a buffer is passed over the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), or determining binding by monitoring changes in the spectral or optical properties of proteins by fluorescence, UV absorbance, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary analyses include, but are not limited to, Western blot, ELISA, analytical ultracentrifugation, spectroscopy, and surface plasmon resonance (Biacore®) analysis (see, e.g., Scatchard et al., Ann. NY Acad. Sci. 51 : 660, 1949; Wilson, Science 295 : 2103, 2002; Wolff et al., Cancer Res. 53 : 2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, or equivalents), flow cytometry, sequencing, and other methods for detecting expressed nucleic acids. In one example, the apparent affinity of a TCR is measured by assessing binding to various concentrations of tetramers, e.g., by flow cytometry, using labeled tetramers. In one example, the apparent _K of a TCR is measured using 2-fold dilutions of labeled tetramer over a range of concentrations, the binding curves are then fit by nonlinear regression, and the apparent _K is determined as the concentration of ligand that gives half-maximal binding.

In some embodiments, the binding molecule exhibits a binding preference for antigenic recognition of cells expressing HPV 16 E6 or E7, as known and/or described herein as specific cells expressing HPV 16 E6 or E7 and known not to express HPV 16 E6 or E7, as compared to HPV 16 E6 or E7 negative cells. In some embodiments, the binding preference is observed when a significantly greater degree of binding is measured to cells expressing HPV 16 E6 or E7 as compared to non-HPV 16 E6 or E7 expressing cells. In some embodiments, the fold change in the extent of binding detected to cells expressing HPV 16 E6 or E7 compared to non-HPV 16 E6 or E7 expressing cells, e.g., as measured by mean fluorescence intensity and/or dissociation constant or EC ₅₀ in a flow cytometry-based assay, is at least or about 1.5, 2, 3, 4, 5, 6, or greater.

In some embodiments, the binding molecules, such as TCRs, do not exhibit cross-reactivity or off-target binding, such as undesirable off-target binding, such as off-target binding to antigens present in healthy or normal tissues or cells. In some embodiments, the binding molecules, such as TCRs, recognize, such as specifically bind, only one peptide antigenic determinant or antigenic complex, such as recognizing only a specific HPV 16 E6 antigenic determinant shown in SEQ ID NO: 268 or an E7 antigenic determinant shown in any one of SEQ ID NO: 271, or an antigenic complex thereof. Thus, in some embodiments, the provided binding molecules, such as TCRs, have a reduced risk of causing undesirable side effects due to, for example, recognition of non-target peptide antigenic determinants.

In some embodiments, a binding molecule, such as a TCR, does not recognize, such as does not specifically bind, a peptide epitope that is sequence-related to the HPV 16 E6 epitope shown in SEQ ID NO: 268 or the E7 epitope shown in SEQ ID NO: 271, i.e., does not recognize an epitope that shares some of the same amino acids as the HPV 16 E6 or E7 epitope shown in either of SEQ ID NOs: 268 or 271, respectively, such as does not recognize an epitope that differs from such an epitope by 1, 2, 3, 4, 5, or 6 amino acid residues when the epitopes are aligned. In some embodiments, the binding molecule, such as a TCR, does not recognize a sequence-unrelated epitope of the HPV 16 E6 shown in SEQ ID NO: 268 or the E7 epitope shown in SEQ ID NO: 271, i.e., does not recognize an epitope that is substantially different in sequence from the HPV 16 E6 or E7 epitope shown in SEQ ID NO: 268 or 271, respectively, such as differing from such epitopes by more than 6, 7, 8, 9, 10 or more amino acid residues when the epitopes are aligned. In some embodiments, the binding molecule, such as a TCR, does not recognize the HPV 16 E6 epitope shown in SEQ ID NO: 268 or the E7 epitope shown in SEQ ID NO: 271 in the context of a different MHC allele, such as in the context of an MHC allele other than HLA-A2.

Typically, specific binding of a binding molecule, such as a TCR, to a peptide antigenic determinant, such as in complex with an MHC, is controlled by the presence of an antigenic binding site containing one or more complementary determining regions (CDRs). In general, it is understood that specific binding does not mean that a particular peptide antigenic determinant, such as in complex with an MHC, is the only antigenic determinant to which an MHC-peptide molecule can bind, as non-specific binding interactions with other molecules may also occur. In some embodiments, the binding affinity of a binding molecule to a peptide in the context of an MHC molecule is higher than binding to such other molecules, such as another peptide in the context of an MHC molecule or an unrelated (control) peptide in the context of an MHC molecule, such as at least about 2 times, at least about 10 times, at least about 20 times, at least about 50 times, or at least about 100 times higher than the binding affinity to such other molecules.

In some embodiments, the safety or off-target binding activity of a binding molecule such as a TCR can be assessed using any of a variety of screening assays known in the art. In some embodiments, the generation of an immune response to a specific binding molecule such as a TCR can be measured in the presence of cells known not to express the target peptide antigenic determinant, such as cells derived from normal tissue, allogeneic cell lines expressing one or more different MHC types or other tissue or cell sources. In some embodiments, the cells or tissues include normal cells or tissues. For example, in some cases, the cell or tissue can include brain, muscle, liver, colon, kidney, lung, ovary, placenta, heart, pancreas, prostate, epithelium or skin, testis, adrenal gland, intestine, bone marrow, or spleen. In some embodiments, binding to cells can be tested in 2-dimensional culture. In some embodiments, binding to cells can be tested in 3-dimensional culture. In some embodiments, as a control, the tissue or cell can be a tissue or cell known to express the target antigenic determinant. Immune responses can be assessed directly or indirectly, such as by assessing activation of immune cells such as T cells (e.g., cytotoxic activity), production of interleukins (e.g., interferon gamma), or activation of signaling cascades.

In some embodiments, potential off-targets can be identified by homology scanning of the human genome using a specific target epitope, for example to identify potential sequence-related epitopes. In some cases, protein sequence databases can be analyzed to identify peptides similar to the target peptide epitope. In some embodiments, to facilitate the identification of potential sequence-related epitopes of interest, binding motifs can be first identified. In some embodiments, the binding motif can be identified by peptide scanning of the target antigenic determinant (e.g., the HPV 16 E6 or E7 antigenic determinant shown in SEQ ID NO: 268 or 271), such as alanine mutation induction scanning, to identify the binding motif recognized by the binding molecule, see, for example, WO2014/096803. In some embodiments, the binding motif can be identified by mutation induction of the target peptide so that a series of mutants are generated, in which each amino acid or a subset thereof is changed to another amino acid residue, and its activity is tested relative to the original target antigenic determinant, and those residues involved in or required for binding are identified. In some embodiments, a series of mutants in which the amino acid residue at each position of the target antigenic determinant is mutated to all alternative amino acids can be obtained. In some cases, once a binding motif (i.e., an amino acid residue that is not tolerated and is involved in or required for binding) is identified, a protein database can be searched for proteins containing the binding motif.

In some embodiments, suitable protein databases include, but are not limited to, UniProtKB/Swiss-Prot (http://www.uniprot.org/), Protein Information Resource (PIR) (http://pir.georgetown.edu/pirwww/index.shtml), and/or Reference Sequence (RefSeq) (www.ncbi.nlm.nih.gov/RefSeq). Peptide motif searches can be performed using any of a variety of tools that can be found on bioinformatics resource websites such as ExPASY (http://www.expasy.org/). For example, the search tool ScanProsite identifies user-defined motifs in all protein sequences in the UniProtKB/Swiss-Prot Protein Knowledgebase (De Castro et al. Nucleic Acids Res. 2006 Jul 1;34(web server issue):W362-5). In some cases, searches may be performed for human-derived peptides or peptides from organisms that are normally found in the human body, such as viruses or bacterial pathogens or commensal bacteria.

In some embodiments, if a potential off-target epitope is identified, a binding molecule such as a TCR can be redesigned so that there is no longer any cross-reactivity with the off-target peptide, while retaining binding to the target peptide epitope, preferably with high affinity. For example, a T cell receptor can be redesigned by mutation induction using the methods described in WO 03/020763.

In some embodiments, binding molecules, such as engineered cells comprising binding molecules such as TCRs, elicit an immune response to HPV 16. In some embodiments, cytotoxic T lymphocytes (CTLs) are activated when cells containing binding molecules such as TCRs come into contact with target cells, such as cancer cells, cells infected with HPV or containing HPV DNA sequences, and/or cells expressing HPV 16, such as HPV 16 E6 or HPV 16 E7. For example, a cell containing a TCR exhibits cytotoxic activity if it induces lysis of a target cell, such as a cancer cell or a cell known to express HPV or contain an HPV DNA sequence, including a cell expressing HPV 16, such as a cell expressing HPV 16 E6 or E7. In some cases, cytotoxic activity can be assessed by incubating or contacting the exemplary cell line SCC152 with T cells expressing a provided binding molecule, such as any of the provided TCRs or antigen-binding fragments thereof.

In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as a cytotoxicity assay or CTL activation, such as described, for example, in Kochenderfer et al., J. Immunotherapy, 32(7):689-702 (2009); and Herman et al., J. Immunological Methods, 285(1):25-40 (2004). There are a variety of techniques for analyzing the activity of CTLs. In some embodiments, CTL activity can be assessed by analyzing the culture for the presence of CTLs that lyse radiolabeled target cells, such as a specific peptide pulsed target. Such techniques include labeling target cells with radionuclides such as Na ₂ , ⁵¹ CrO ₄ or ³ H-thymidine, and measuring the release or retention of the radionuclides from the target cells as an index of cell death. In some embodiments, CTLs are known to release a variety of interleukins when they are stimulated by appropriate target cells, such as tumor cells expressing relevant MHC molecules and corresponding peptide antigenic determinants, and the presence of such antigenic determinant-specific CTLs can be determined by measuring interleukin release. Non-limiting examples of such interleukins include IFN-γ, TNF-α, and GM-CSF. Analysis of such interleukins is well known in the art, and its selection is left to those skilled in the art. Coligan, JE et al. (Current Protocols in Immunology, 1999, John Wiley & Sons, Inc., New York) provide methods for measuring target cell death and interleukin release as measures of CTL responsiveness.

In some aspects, the ability of a binding molecule, such as a cell expressing the binding molecule (e.g., a TCR or CAR), to elicit an immune response can be determined by measuring interleukin release. In some embodiments, in response to co-culture with or exposure to cells expressing the binding molecule (e.g., a TCR or CAR), a variety of interleukins are released when the cells are stimulated by appropriate target cells known to express HPV 16, such as HPV 16 E6 or HPV 16 E7. Non-limiting examples of such interleukins include IFN-γ, TNF-α, and GM-CSF. In some embodiments, interleukin production can be measured as an indication of an immune response. In some cases, such interleukins measured may include, but are not limited to, interleukin-2 (IL-2), interferon-γ (IFNγ), interleukin-4 (IL-4), TNF-α, interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), or TGF-β. Assays for measuring interleukins are well known in the art and include, but are not limited to, ELISA, intracellular interleukin staining, cytological bead arrays, RT-PCR, ELISPOT, flow cytometry, and bioassays in which the responsiveness (e.g., proliferation) of cells in response to the relevant interleukin is tested in the presence of a test sample.

Exemplary cells known to express HPV 16 include, but are not limited to, CaSki cells (ATCC No. CRL-1550, which contain approximately 600 copies of integrated HPV 16), SCC152, or other tumor cells expressing relevant MHC molecules and corresponding peptide epitopes, such as HPV 16 E6 or E7 epitopes, such as any of the epitopes shown in SEQ ID NO: 268 or 271.

In some embodiments, cells exposed to binding molecules, such as cells containing binding molecules (such as TCR or CAR), are assessed for immune readout, such as using T cell analysis. In some embodiments, cells containing binding molecules can activate CD8+ T cell responses. In one embodiment, CD8+ T cell responses can be assessed by monitoring CTL responsiveness using assays, including (but not limited to) target cell lysis or detection of interferon gamma release via ⁵¹ Cr release, such as by enzyme-linked immunosorbent dot assay (ELISA), intracellular interleukin staining, or ELISPOT. In some embodiments, binding molecules, such as cells containing binding molecules such as TCR or CAR, can activate CD4+ T cell responses. In some aspects, CD4+ T cell responses can be assessed by an assay that measures proliferation, such as by incorporation of [3H]-thymidine into cellular DNA and/or by the production of interleukins, such as by ELISA, intracellular interleukin staining, or ELISPOT. In some cases, the interleukin may include, for example, interleukin-2 (IL-2), interferon-γ (IFN-γ), interleukin-4 (IL-4), TNF-α, interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), or TGFβ. In some embodiments, recognition or binding of a peptide epitope, such as an MHC class II epitope, by a binding molecule can elicit or activate a CD4+ T cell response and/or a CD8+ T cell response.

In some embodiments, the binding specificity and/or function (e.g., the ability to elicit an immune response to HPV 16) of a binding molecule, such as a TCR or antigen-binding fragment thereof, is at least partially CD8-independent. In some cases, TCR recognition of a peptide in the context of an MHC molecule and subsequent T cell activation is promoted in the presence of a CD8 co-receptor. For example, engagement of a CD8 co-receptor can promote low to moderate TCR affinity interactions and/or T cell activation (see, e.g., Kerry et al. J. Immunology (2003) 171(9): 4493-4503; and Robbins et al. J Immunology (2008) 180(9): 6116-6131). The binding molecules provided are molecules, such as TCRs, that exhibit CD8-independent binding to HPV E6 or E7 peptide epitopes. Exemplary such TCRs are TCRs designated as TCR 57 as described herein or TCRs containing the CDRs of a TCR designated as TCR 57 and/or Vα or Vβ chains of such TCRs, see, e.g., Tables 5, 6, and 7. In some embodiments, such binding molecules, such as TCRs, can have a higher functional affinity or avidity than TCRs or antigen-binding fragments thereof that require the presence of CD8 co-expression. In some aspects, the CD8-independent binding molecules provided, such as TCRs, can be expressed or engineered in cells that do not express CD8 (e.g., T cells), such as can be expressed or engineered in CD4+ cells. In some embodiments, the engineered non-CD8 expressing cells provided, such as CD4+ cells, are cells that express recombinant binding molecules, such as TCRs or antigen binding fragments, that exhibit binding specificity, affinity, and/or avidity for a peptide in the context of an MHC molecule that is at least or at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater than the same binding molecule (e.g., TCR or antigen binding fragment thereof) expressed on CD8+ T cells.

III. Methods for Identifying and Producing T Cell Receptors

In some embodiments, methods for identifying and generating T cell receptors for target antigens are provided. In some aspects, the methods include subjecting a biological sample containing T cells, such as primary T cells, including T cells derived from a normal donor or a patient with a disease or condition of interest, to multiple rounds of antigen exposure and assessment. In some aspects, each round includes the use of artificial or engineered antigen presenting cells, such as autologous dendritic cells or other APCs pulsed with the desired peptide antigen to promote presentation on MHC, such as class I or class II MHC. In some aspects, multiple rounds of antigen exposure are performed and in some aspects, T cells are sorted after one or more rounds, for example based on the ability to bind to the desired antigen (such as a peptide-MHC tetramer). In some aspects, sorting is performed by flow cytometry. In some aspects, cells that are thought to bind to a desired antigen (positive fraction) and cells that are thought not to bind to the antigen are assessed, for example, by single cell sequencing. In some aspects, the methods sequence and identify TCR pairs present in each sample at the single cell level. In some aspects, the methods can quantify the number of copies of a given TCR pair present in a sample, and thus can assess the abundance of a given TCR in a given sample and/or its enrichment relative to another sample, such as enrichment or abundance in the positive (antigen binding) fraction, for example, over one or more rounds, for example compared to the negative fraction. In some embodiments, such assays are performed to generate antigen-specific T cell receptors (TCRs) that specifically bind to human papillomavirus 16 or 18 peptide antigens, such as peptides derived from E6 or E7, such as E6 (29-38) or E7 (11-19) peptides, for example, that are presented on MHC-I molecules and survive and/or are enriched over time after multiple rounds of antigen stimulation. In some embodiments, pure T cell lines are generated and the sequences of individual paired TCR alpha and beta chains and their abundance in various populations are determined on a single cell basis using high-throughput paired TCR sequencing.

In some aspects, peptide-pulsed HLA:A02:01APCs are generated with HPV 16 E6(29-38) peptide (TIHDIILECV; SEQ ID NO: 268) or E7(11-19) peptide (YMLDLQPET; SEQ ID NO: 271). Autologous CD8+ T cells from normal human donors are cultured with the peptide-pulsed cells for multiple rounds and selected based on binding to peptide-loaded autologous MHC tetramers.

In some aspects, cells are subjected to multiple rounds of stimulation in the presence of peptide pulsed cells, such as a total of two or three or more rounds of stimulation (e.g., three rounds maintaining a specific peptide concentration of 1000 ng/mL). After one or more rounds of stimulation, such as after the first round of stimulation and/or after the second and third rounds of stimulation, the cells are sorted by flow cytometry into positive and negative populations for binding to peptide-MHC tetramers containing the appropriate tetramer, respectively. In some embodiments, cells from tetramer-positive and -negative populations undergo single cell TCR sequencing at each of one or more rounds or after one or more rounds, such as the second and third rounds, to assess the presence and frequency of individual TCRs in the different populations, and the persistence of TCR homologues during multiple rounds of antigen stimulation.

In some aspects, cell populations from the positive and negative fractions (i.e., sorted by flow cytometry based on positive and negative staining, respectively, for binding to an antigen of interest, such as a peptide-MHC, such as a loaded tetramer, e.g., as determined by flow cytometry) are subjected to high-throughput single-cell sequencing for TCR alpha and beta chain pairs after one or more rounds. In some aspects, high-throughput single-cell TCR sequencing is performed as generally described in published PCT patent application publications Nos. WO2012/048340, WO2012/048341, and WO2016/044227. Thus, in some aspects, sequencing methods employ single cell droplets and samples and molecular barcodes to identify individual pairs of TCR alpha and beta chain sequences at the single cell level for each of a large number (e.g., millions) of single cells present in a single starting composition, and to assess the abundance of each TCR pair in each population assessed. In some embodiments, the ability to identify and quantify TCR pairs at the single cell level allows for the assessment of the frequency of each of each TCR pair in each of the individual positive and negative fractions, and the assessment of the enrichment and persistence of TCRs during multiple rounds of antigen stimulation.

In some aspects, the methods generate, identify, separate and/or select TCR pairs that are enriched in antigen-binding (e.g., peptide-binding) moieties after at least one, and in some aspects, multiple, rounds of stimulation. In some aspects, the TCRs are present and/or present in a desired abundance and/or preferentially enriched after round 1, round 2, and/or round 3, and in some aspects, at least multiple rounds of antigen exposure. In some aspects, after multiple rounds of antigen exposure, the TCRs in a population are enriched over time. TCRs generated or identified using such methods are also provided, such as TCRs having such properties, such as the ability to survive and/or proliferate during multiple rounds of antigen exposure, such as in an analysis of APCs pulsed with peptides.

IV. Engineered cells

Cells are also provided, such as cells that have been engineered to contain a binding molecule described herein. Populations of such cells, compositions containing such cells and/or enriched for such cells are also provided, such as where cells expressing the binding molecule account for at least 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the total cells in the composition, or certain types of cells, such as T cells or CD8+ or CD4+ cells. In some embodiments, the cells are primary T cells. The compositions are pharmaceutical compositions and formulations for administration, such as for administering cell therapy. Also provided are therapeutic methods for administering cells and compositions to an individual (e.g., a patient).

Therefore, genetically engineered cells expressing binding molecules are also provided. The cells are generally eukaryotic cells, such as mammalian cells, and are usually human cells. In some embodiments, the cells are derived from blood, bone marrow, lymph or lymphoid organs, and are immune system cells, such as innate or acquired immune cells, such as bone marrow or lymphocytes, including lymphocytes, usually T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells are typically primary cells, such as cells isolated directly from an individual and/or isolated from an individual and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as complete T cell populations, CD4+ cells, CD8+ cells and subsets thereof, such as those defined according to the following: function, activation state, maturity, differentiation potential, expansion, recirculation, location and/or persistence ability, antigen specificity, antigen receptor type, presence in a specific organ or compartment, marker or cytokine secretion profile and/or degree of differentiation. When referring to the individual being treated, the cells can be allogeneic cells and/or autologous cells. Such methods include existing methods. In some embodiments, such as in the prior art, the cells are pluripotent and/or multipotent cells, such as stem cells, such as induced pluripotent stem cells (iPSC). In some embodiments, the method comprises isolating the cells from an individual, preparing, processing, culturing and/or engineering them as described herein, and reintroducing them into the same patient before or after cryopreservation.

Subtypes and subpopulations of T cells and/or CD4+ T cells and/or CD8+ T cells include naive T cells ( _TN ), effector T cells ( _TEFF ), memory T cells and their subtypes, such as stem cell memory T cells ( _TSCM ), central memory T cells ( _TCM ), effector memory T cells ( _TEM) , and so on. ) or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α/β T cells and δ/γ T cells.

In some embodiments, the cell is a natural killer (NK) cell. In some embodiments, the cell is a monocyte or a granulocyte, such as a bone marrow cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and/or a basophil.

In some embodiments, the cell includes one or more nucleic acids introduced by genetic engineering and thereby expresses a recombinant or genetically engineered product of such nucleic acids. In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or in a sample obtained from the cell, such as a nucleic acid obtained from another organism or cell, e.g., not normally present in the engineered cell and/or the organism from which such cell is derived. In some embodiments, the nucleic acid is non-naturally occurring, such as a nucleic acid not found in nature, including nucleic acids comprising a chimeric combination of nucleic acids encoding different domains from a variety of different cell types.

In some embodiments, the genes and/or gene products (and/or their expression) in the provided cells and/or compositions containing such cells are reduced, deleted, eliminated, knocked out or interrupted. In some aspects, such genes and/or gene products include one or more of the genes (or their products) encoding TCR α (TRAC) and/or TCR β (TRBC), for example to reduce or prevent the expression of endogenous TCRs and/or their chains in cells, such as T cells. In some embodiments, reducing or preventing the expression of endogenous TCRs can result in a reduced risk or likelihood of mismatches between the engineered TCR and the chains of the endogenous TCR, thereby generating new TCRs that may result in a higher risk of undesirable or unexpected antigen recognition and/or side effects, and/or may reduce the amount of expression of a desired exogenous TCR. In some aspects, reducing or preventing endogenous TCR expression can increase the expression of the engineered TCR in cells, such as by 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or more, compared to cells in which TCR expression is not reduced or prevented. For example, in some cases, suboptimal expression of the engineered or recombinant TCR may occur due to competition with the endogenous TCR and/or with TCRs with mismatched linkages for participation in the invariant CD3 signaling molecule that allows the complex to be expressed on the cell surface. In some embodiments, reduction, deletion, elimination, gene knockout or interruption is performed by gene editing, such as using zinc finger nucleases (ZFNs), TALENs, or CRISPR/Cas systems with engineered single guide RNAs (gRNAs) that cut the TCR gene. In some embodiments, reducing the expression of endogenous TCRs is performed using inhibitory nucleic acid molecules directed against target nucleic acids encoding specific TCRs (e.g., TCR-α and TCR-β). In some embodiments, the inhibitory nucleic acid is or contains or encodes a small interfering RNA (siRNA), a microRNA-adapted shRNA, a short hairpin RNA (shRNA), a hairpin siRNA, a microRNA (miRNA-precursor), or a microRNA (miRNA). Exemplary methods for reducing or preventing the expression of endogenous TCRs are known in the art, see, for example, U.S. Patent No. 9,273,283; U.S. Publication No. US2014/0301990; and PCT Publication No. WO2015/161276.

A. Preparation of cells for genetic engineering

In some embodiments, the preparation of engineered cells includes one or more culture and/or preparation steps. Cells for introduction of binding molecules such as TCRs or CARs can be isolated from a sample, such as a biological sample, such as a biological sample obtained or derived from an individual. In some embodiments, the individual from whom the cells are isolated is an individual suffering from a disease or condition or in need of cell therapy or to be administered cell therapy. In some embodiments, the individual is a human in need of a specific therapeutic intervention, such as a donor-receiver cell therapy in which cells are isolated, processed and/or engineered.

Thus, in some embodiments, the cells are primary cells, such as primary human cells. Samples include tissues, body fluids, and other samples obtained directly from an individual, as well as samples produced by one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with a viral vector), washing, and/or culturing. Biological samples can be samples obtained directly from a biological source or processed samples. Biological samples include (but are not limited to) body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat; tissue and organ samples, including processed samples obtained therefrom.

In some aspects, the sample from which the cells are derived or from which the cells are isolated is a blood sample or a blood-derived sample or is or is derived from a blood apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), white blood cells, bone marrow, thymus, tissue sections, tumors, leukemias, lymphomas, lymph nodes, intestinal-associated lymphoid tissue, mucosal-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testicles, ovaries, tonsils or other organs, and/or cells obtained therefrom. In the case of cell therapy, such as donor-transfer cell therapy, samples include samples of autologous and allogeneic origin.

In some embodiments, the cells are derived from a cell line, such as a T cell line. In some embodiments, the cells are obtained from a xenogeneic source, such as from a mouse, rat, non-human primate, and pig.

In some embodiments, the isolation of cells includes one or more preparation steps and/or non-affinity-based cell isolation steps. In some embodiments, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove undesirable components, enrich for desired components, solubilize, or remove cells sensitive to a particular reagent. In some embodiments, cells are isolated based on one or more properties, such as density, adhesion properties, size, sensitivity, and/or resistance to a particular component.

In some instances, cells are obtained from the individual's circulating blood, such as by hemopheresis or leukapheresis. In some embodiments, the sample contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some embodiments contains cells other than red blood cells and platelets.

In some embodiments, blood cells collected from an individual are washed, for example to remove the plasma fraction and the cells are placed in an appropriate buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution does not contain calcium and/or magnesium and/or multiple or all divalent cations. In some aspects, the washing step is performed using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the washing step is performed by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers (such as PBS without Ca ⁺⁺ /Mg ⁺⁺ ) after washing. In certain embodiments, components of the blood sample are removed and the cells are directly resuspended in culture medium.

In some embodiments, the method includes density-based cell separation methods, such as preparing white blood cells from peripheral blood by lysing red blood cells and centrifugation through Percoll or Ficoll gradients.

In some embodiments, the separation method includes separating different cell types based on the expression or presence of one or more specific molecules, such as surface markers (e.g., surface proteins), intracellular markers, or nucleic acids in the cell. In some embodiments, any known separation method based on such markers can be used. In some embodiments, the separation is based on affinity or immunoaffinity. For example, in some embodiments, separation includes separating cells and cell populations based on the cell's expression or amount of expression of one or more markers, typically cell surface markers, such as by incubation with antibodies or binding partners that specifically bind to such markers, followed by typically a wash step and separation of cells that have bound the antibody or binding partner from cells that have not bound the antibody or binding partner.

Such separation steps can be based on positive selection, in which cells that have bound the reagent are retained for further use; and/or negative selection, in which cells that have not bound the antibody or binding partner are retained. In some instances, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where antibodies are not available that specifically identify cell types in a heterogeneous population, and therefore separation based on markers expressed by cells other than the desired population is optimal.

Isolation does not require 100% enrichment or depletion of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell (e.g., cells expressing a marker) means increasing the number or percentage of such cells without necessarily eliminating cells that do not express the marker. Similarly, negative selection, depletion, or depletion of a particular type of cell (e.g., cells expressing a marker) means decreasing the number or percentage of such cells without necessarily eliminating all such cells.

In some examples, multiple rounds of separation steps are performed, wherein the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can be performed to simultaneously express cells for multiple markers, such as by incubating the cells with multiple antibodies or binding partners that are each specific for the marker targeted by the negative selection. Similarly, multiple cell types can be simultaneously positively selected by incubating the cells with multiple antibodies or binding partners expressed on the various cell types.

For example, in some aspects, specific subsets of T cells, such as cells that are positive or express large amounts of one or more surface markers, such as CD28 ⁺ , CD62L ⁺ , CCR7 ⁺ , CD27 ⁺ , CD127 ⁺ , CD4 ⁺ , CD8 ⁺ , CD45RA ⁺ and/or CD45RO ⁺ T cells, are isolated by positive or negative selection techniques.

For example, CD3 ⁺ , CD28 ⁺ T cells can be positively selected using anti-CD3/anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).

In some embodiments, separation is performed by enriching a specific cell population by positive selection or depleting a specific cell population by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers that are expressed or expressed in relatively high amounts (marker ^high ) (marker ⁺ ) on the positively or negatively selected cells, respectively.

In some embodiments, T cells are isolated from PBMC samples by negative selection for markers expressed on non-T cells, such as B cells, monocytes or other white blood cells (such as CD14). In some aspects, CD4 ⁺ helper and CD8 ⁺ cytotoxic T cells are isolated using a CD4 ⁺ or CD8 ⁺ selection step. Such CD4 ⁺ and CD8 ⁺ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively high degree on one or more naive, memory and/or effector T cell subsets.

In some embodiments, CD8 ⁺ cells are further enriched or depleted for naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subsets. In some embodiments, central memory T ( _TCM ) cells are enriched to improve efficacy, such as improving long-term survival, expansion, and/or engraftment after administration, which in some aspects is particularly robust in this subset. See Terakura et al. (2012) Blood. 1: 72-82; Wang et al. (2012) J Immunother. 35(9): 689-701. In some embodiments, combining _TCM -enriched CD8 ⁺ T cells with CD4 ⁺ T cells can further enhance efficacy.

In an embodiment, memory T cells are present in CD62L ⁺ and ^CD62L- subsets of CD8 ⁺ peripheral blood lymphocytes. For example, anti-CD8 and anti-CD62L antibodies can be used to enrich or deplete the CD62L ^- CD8 ⁺ and/or CD62L ⁺ CD8 ⁺ fractions of PBMCs.

In some embodiments, enrichment of central memory T ( _TCM ) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3 and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, a CD8 ⁺ population enriched in _TCM cells is isolated by depletion of cells expressing CD4, CD14, CD45RA and positive selection or enrichment for cells expressing CD62L. In one embodiment, the enrichment of central memory T ( _TCM ) cells begins with a negative cell fraction selected based on CD4 expression, which is negatively selected based on the expression of CD14 and CD45RA, and positively selected based on CD62L. Such selections are performed simultaneously in some embodiments and sequentially in any order in other embodiments. In some embodiments, the same selection step based on CD4 expression used to prepare the CD8 ⁺ cell population or subset is also used to generate the CD4 ⁺ cell population or subset, thereby retaining the positive and negative fractions from the CD4-based separation and using them in subsequent steps of the method, optionally after one or more additional positive or negative selection steps.

In one embodiment, a PBMC sample or other white blood cell sample is subjected to selection for CD4 ⁺ cells, wherein both the negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on the expression of CD14 and CD45RA, and positive selection is performed based on markers specific to central memory T cells, such as CD62L or CCR7, wherein the positive and negative selections are performed in either order.

CD4 ⁺ T helper cells are sorted into naive cells, central memory cells and effector cells by identifying cell populations with cell surface antigens. CD4 ⁺ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4 ⁺ T lymphocytes are CD45RO ^- , CD45RA ⁺ , CD62L ⁺ , CD4 ⁺ T cells. In some embodiments, central memory CD4 ⁺ cells are CD62L ⁺ and CD45RO ⁺ . In some embodiments, effector CD4 ⁺ cells are CD62L ^- and CD45RO ^- .

In one example, to enrich CD4 ⁺ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding partners are bound to a solid support or matrix such as magnetic beads or paramagnetic beads to isolate cells for positive and/or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in Methods in Molecular Medicine, Volume 58: Metastasis Research Protocols, Volume 2: Cell Behavior In Vitro and In Vivo, pages 17-25, edited by SA Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).

In some embodiments, a sample or composition of cells to be separated is incubated with a small magnetizable or magnetically responsive material, such as a magnetically responsive particle or microparticle, such as a paramagnetic bead (e.g., Dynal beads or MACS beads). The magnetically responsive material, such as a particle, is typically directly or indirectly linked to a binding partner, such as an antibody, which specifically binds to a molecule, such as a surface marker, present on the cell, multiple cells, or cell population desired to be separated, such as desired to be negatively or positively selected.

In some embodiments, the magnetic particles or beads comprise a magnetically reactive material bound to a specific binding member such as an antibody or other binding partner. There are a variety of well-known magnetically reactive materials that can be used in magnetic separation methods. Suitable magnetic particles include those described in U.S. Patent No. 4,452,773 to Molday and European Patent Specification EP 452342 B, which are incorporated herein by reference. Colloidal-sized particles, such as those described in U.S. Patent No. 4,795,698 to Owen and U.S. Patent No. 5,200,084 to Liberti et al., are other examples.

The incubation is generally carried out under conditions where the antibody or binding partner linked to the magnetic particles or beads or a molecule that specifically binds to such an antibody or binding partner (such as a secondary antibody or other reagent) specifically binds to the cell surface molecule (if present) on the cells in the sample.

In some embodiments, the sample is placed in a magnetic field and those cells to which magnetically responsive or magnetizable particles are attached are attracted to the magnet and separated from unlabeled cells. For positive selection, cells attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some embodiments, a combination of positive and negative selections is performed during the same selection step, wherein both the positive and negative fractions are retained and further processed or subjected to a further separation step.

In some embodiments, magnetic reactive particles are coated with primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In some embodiments, magnetic particles are linked to cells via coating with primary antibodies specific for one or more markers. In some embodiments, cells rather than beads are labeled with primary antibodies or binding partners, and magnetic particles coated with cell type specific secondary antibodies or other binding partners (e.g., streptavidin) are then added. In some embodiments, streptavidin-coated magnetic particles are used in conjunction with primary or secondary antibodies labeled with biotin.

In some embodiments, the magnetically reactive particles remain attached to cells that are subsequently cultured, grown, and/or engineered; in some aspects, the particles remain attached to cells that are administered to a patient. In some embodiments, the magnetizable or magnetically reactive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of unlabeled competitive antibodies, magnetizable particles, or antibodies bound to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

In some embodiments, affinity-based selection is performed by magnetic-activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetic-activated cell sorting (MACS) systems are capable of selecting cells attached to magnetized particles with high purity. In some embodiments, MACS is operated in a mode that sequentially dissolves non-target and target species after applying an external magnetic field. That is, cells attached to the magnetized particles remain in place, while unattached species are dissolved. Then, after this first dissolution step is completed, species that are trapped in the magnetic field and cannot be dissolved are released in some manner, allowing them to be dissolved and recovered. In some embodiments, non-target cells are labeled and depleted from the heterogeneous cell population.

In certain embodiments, isolation or separation is performed using a system, device or apparatus that performs one or more of the isolation, cell preparation, separation, processing, cultivation, culturing and/or formulation steps of the methods. In some aspects, the system is used to perform each of these steps in a closed or sterile environment, for example to minimize errors, user manipulation and/or contamination. In one example, the system is a system as described in International Patent Application Publication No. WO2009/072003 or US 2011/0003380 A1.

In some embodiments, the system or device performs one or more, such as all, of the separation, processing, engineering, and formulation steps in an integrated or self-contained system and/or in an automated or programmable manner. In some aspects, the system or device includes a computer and/or a computer program that communicates with the system or device, which allows a user to program, control, evaluate the results of, and/or adjust various aspects of the processing, separation, engineering, and formulation steps.

In some embodiments, separation and/or other steps are performed using a CliniMACS system (Miltenyi Biotec), for example for automated separation of cells on a clinical scale in a closed and sterile system. Components may include an integrated microcomputer, a magnetic separation unit, a peristaltic pump, and various pinch valves. In some embodiments, the integrated computer controls all components of the instrument and guides the system to perform repetitive procedures in a standardized sequence. In some embodiments, the magnetic separation unit includes a movable permanent magnet and a holder for selecting the tubing column. The peristaltic pump controls the flow rate through the entire tubing set and together with the pinch valves ensures controlled flow of the buffer solution through the system and continuous suspension of the cells.

In some aspects, the CliniMACS system uses magnetizable particles coupled to antibodies supplied in a sterile, non-pyrolyzed solution. In some embodiments, after the cells are labeled with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to the tube set, which is in turn connected to a bag containing a buffer and a cell collection bag. The tube set consists of pre-assembled sterile tubes, including a front column and a separation column, and is for single use only. After the separation program is initiated, the system automatically applies the cell sample to the separation column. The labeled cells are retained in the column, and the unlabeled cells are removed by a series of washing steps. In some embodiments, the cell population suitable for the methods described herein is not labeled and is not retained in the column. In some embodiments, a cell population suitable for use in the methods described herein is labeled and retained in a column. In some embodiments, after the magnetic field is removed, the cell population suitable for use in the methods described herein is eluted from the column and collected in a cell collection bag.

In certain embodiments, separation and/or other steps are performed using a CliniMACS Prodigy system (Miltenyi Biotec). In some aspects, the CliniMACS Prodigy system is equipped with a cell processing unit that allows automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system may also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by identifying macroscopic layers of source cell products. For example, peripheral blood can be automatically separated into red blood cells, white blood cells, and a plasma layer. The CliniMACS Prodigy system may also include an integrated cell culture chamber to enable cell culture protocols such as cell differentiation and expansion, antigen loading, and long-term cell culture. The input port allows sterile removal and replenishment of the culture medium and monitoring of cells using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660; Terakuraet et al. (2012) Blood. 1: 72-82; and Wang et al. (2012) J Immunother. 35(9): 689-701.

In some embodiments, the cell populations described herein are collected and enriched (or depleted) by flow cytometry, in which cells stained with a variety of cell surface markers are carried in a fluid stream. In some embodiments, the cell populations described herein are collected and enriched (or depleted) by preparative (FACS) sorting. In certain embodiments, the cell populations described herein are collected and enriched (or depleted) by using a combination of a microelectromechanical system (MEMS) chip and a FACS-based detection system (see, e.g., WO 2010/033140; Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5): 355-376). In both cases, cells can be labeled with a variety of markers, allowing the isolation of well-defined T cell subsets with high purity.

In some embodiments, antibodies or binding partners are labeled with one or more detectable markers to facilitate separation for positive and/or negative selection. For example, separation can be based on binding to fluorescently labeled antibodies. In some embodiments, cell separation based on binding of antibodies or other binding partners specific for one or more cell surface markers is performed in a fluid stream, such as by fluorescence activated cell sorting (FACS), including preparative (FACS) and/or microelectromechanical systems (MEMS) chips, such as in combination with a flow cytometry detection system. Such methods allow for simultaneous positive and negative selection based on multiple markers.

In some embodiments, the preparation method includes a step of freezing (e.g., cryopreserving) the cells before or after isolation, cultivation, and/or engineering. In some embodiments, the freezing and subsequent thawing steps remove granulocytes and, to a certain extent, monocytes from the cell population. In some embodiments, the cells are suspended in a freezing solution, such as after a washing step, to remove plasma and platelets. In some aspects, any of a variety of known freezing solutions and parameters can be used. One example involves the use of PBS or other suitable cell freezing medium containing 20% DMSO and 8% human serum albumin (HSA). It was then diluted 1:1 with culture medium, resulting in final concentrations of DMSO and HSA of 10% and 4%, respectively. The cells were then frozen at a rate of 1° per minute to -80°C and stored in the vapor phase of a liquid nitrogen storage tank.

In some embodiments, the provided methods include cultivation, breeding, culturing and/or genetic engineering steps. For example, in some embodiments, methods for culturing and/or engineering exhausted cell populations and compositions for initiating culture are provided.

Thus, in some embodiments, a cell population is cultured in a composition for initial culture. Cultivation and/or engineering can be performed in a culture vessel, such as a unit, chamber, well, column, tube, tube set, valve, vial, culture dish, bag or other container for culturing or growing cells.

In some embodiments, cells are cultured and/or cultured prior to or in conjunction with genetic engineering. The culture step may include culturing, growing, stimulating, activating and/or propagating. In some embodiments, the composition or cells are cultured in the presence of stimulating conditions or stimulants. Such conditions include those designed to induce proliferation, expansion, activation and/or survival of cells in a population, simulate antigen exposure and/or pre-sensitize cells for genetic engineering (e.g., for the introduction of antigen receptors).

Conditions may include one or more of the following: specific culture medium, temperature, oxygen content, carbon dioxide content, time, agents such as nutrients, amino acids, antibiotics, ions and/or stimulatory factors such as interleukins, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors and any other agents designed to activate cells.

In some embodiments, the stimulating conditions or agents include one or more agents, such as ligands, that are capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent initiates or starts the TCR/CD3 intracellular signaling cascade in the T cell. Such agents may include antibodies, such as antibodies specific for TCR components and/or co-stimulatory receptors, such as anti-CD3. In some embodiments, the stimulating conditions include one or more agents, such as ligands, such as anti-CD28, that are capable of stimulating co-stimulatory receptors. In some embodiments, such agents and/or ligands may be bound to a solid carrier such as a bead and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti-CD28 antibodies to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulator includes IL-2, IL-15 and/or IL-7. In some aspects, the IL-2 concentration is at least about 10 units/ml.

In some embodiments, the cultivation is performed according to techniques such as those described in U.S. Patent No. 6,040,177 to Riddell et al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1: 72-82, and/or Wang et al. (2012) J Immunother. 35(9): 689-701.

In some embodiments, T cells are expanded by adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to the composition of the initial culture (e.g., such that the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells may include gamma-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000 to 3600 rad to prevent cell division. In some embodiments, feeder cells are added to the culture medium prior to adding the T cell population.

In some embodiments, the stimulation conditions include a temperature suitable for the growth of human T lymphocytes, such as at least about 25°C, typically at least about 30°C, and typically at or about 37°C. Optionally, the culture may further include the addition of non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. The LCL may be irradiated with gamma rays in the range of about 6000 to 10,000 rad. In some aspects, the LCL feeder cells are provided in any suitable amount, such as a ratio of LCL feeder cells to naive T lymphocytes of at least about 10:1.

In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen-specific T lymphocytes with an antigen. For example, antigen-specific T cell strains or clones against a cellular giant virus antigen can be generated by isolating T cells from an infected individual and stimulating the cells in vitro with the same antigen.

B. Vectors and methods for genetic engineering

Also provided are methods, nucleic acids, compositions and kits for expressing binding molecules and for producing genetically engineered cells expressing such binding molecules. Genetic engineering generally involves introducing a nucleic acid encoding a binding molecule (e.g., a TCR or CAR, e.g., a TCR-like CAR) into a cell, such as by retroviral transduction, transfection or transformation.

In some embodiments, gene transfer is achieved by first stimulating the cells, such as by combining them with a stimulus that induces a response such as proliferation, survival and/or activation, for example as measured by expression of cytokines or activation markers, and then transducing the activated cells and expanding them in culture to a number sufficient for clinical use.

In some cases, overexpression of stimulatory factors (e.g., lymphokines or interleukins) can be toxic to an individual. Thus, in some cases, the engineered cells include gene segments that render the cells susceptible to negative selection in vivo, such as when administered in a transmissive immunotherapy. For example, in some aspects, the cells are engineered so that they can be eliminated as a result of changes in the in vivo conditions of the patient to whom they are administered. Negatively selectable phenotypes can result from the insertion of genes that confer sensitivity to an administered agent (e.g., a compound). Negatively selectable genes include the herpes simplex virus type I thymidine kinase (HSV-I TK) gene that confers sensitivity to ganciclovir (Wigler et al., Cell 2: 223, 1977); cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89: 33 (1992)).

In some embodiments, the cells are further engineered to promote expression of cytokines or other factors. Various methods for introducing genetically engineered components are well known and can be used with the provided methods and compositions. Exemplary methods include methods for transferring nucleic acids encoding binding molecules, including via viruses (e.g., retroviruses or lentiviruses), transduction, transposons, and electroporation.

In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious viral particles, such as vectors derived from simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors (such as γ-retroviral vectors) (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 Nov 29(11): 550-557).

In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), such as a retroviral vector derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), or spleen focus forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retrovirus includes a retrovirus derived from any avian or mammalian cell source. Retroviruses are generally bitropic, meaning that they are able to infect host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol, and/or env sequences. A variety of illustrative retroviral systems have been described (e.g., U.S. Patent Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A.D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

Lentiviral transduction methods are known. Exemplary methods are described in, for example, Wang et al. (2012) J. Immunother. 35(9):689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506:97-114; and Cavalieri et al. (2003) Blood. 102(2):497-505.

In some embodiments, the recombinant nucleic acid is transferred into T cells via electroporation (see, e.g., Chicaybam et al., (2013) PLoS ONE 8(3):e60298 and Van Tedeloo et al., (2000) Gene Therapy 7(16):1431-1437). In some embodiments, the recombinant nucleic acid is transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4):427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506:115-126). Other methods for introducing genetic material into immune cells and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. NY), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-promoted microprojectile bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

Other methods and vectors for transferring nucleic acids encoding binding molecules or recombinant products are, for example, those described in International Patent Application Publication No. WO2014/055668 and U.S. Patent No. 7,446,190.

Other nucleic acids (e.g., genes) for introduction include those that improve therapeutic efficacy, such as by promoting the viability and/or function of the transferred cells; genes that provide genetic markers for selection and/or assessment of cells, such as to assess survival or localization in vivo; genes that improve safety, for example, by making the cells susceptible to negative selection in vivo, as described by Lupton SD et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also Lupton et al. Publication PCT/US91/08442 and PCT/US94/05601, which describe the use of bifunctional selectable fusion genes obtained by fusion of a dominant positive selectable marker to a negative selectable marker. See, e.g., Riddell et al., U.S. Patent No. 6,040,177, columns 14-17.

Thus, in some embodiments, engineered cells are provided, such as engineered cells containing a binding molecule (such as a TCR or antigen binding fragment thereof or an antibody or antigen binding fragment thereof), a nucleic acid, or a vector as described herein. In some aspects, the cell is produced by transducing a cell in vitro or ex vivo with a vector described herein. In some aspects, the cell is a T cell, such as a CD8+ or CD4+ T cell. In some embodiments, the binding molecule is heterologous to the cell.

V. Compositions, methods and uses

Also provided are compositions including binding molecules such as TCRs and engineered cells, including pharmaceutical compositions and formulations, and methods and uses of such molecules and compositions, such as for the treatment of diseases, conditions and disorders expressing HPV16 E6 or E7, and/or detection, diagnosis and prognosis methods.

A. Pharmaceutical compositions and formulations

Pharmaceutical formulations are provided, which include binding molecules, such as TCRs or antigen-binding fragments thereof or antibodies or antigen-binding fragments thereof, and/or engineered cells expressing binding molecules. Pharmaceutical compositions and formulations generally include one or more pharmaceutically acceptable carriers or excipients, as appropriate. In some embodiments, the composition includes at least one other therapeutic agent.

The term "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective and that contains no additional components that are unacceptably toxic to the subject to which the formulation is to be administered.

"Pharmaceutically acceptable carriers" refer to ingredients in pharmaceutical formulations that are non-toxic to the individual except for the active ingredient. Pharmaceutically acceptable carriers include (but are not limited to) buffers, excipients, stabilizers or preservatives.

In some embodiments, the choice of carrier is determined in part by the specific cell or binding molecule and/or by the method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methyl paraben, propyl paraben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixture thereof is typically present in an amount of about 0.0001% to about 2% of the total composition weight. Carriers are described, for example, by Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexahydroxyquaternary ammonium chloride; benzylammonium chloride; benzethonammonium chloride; butylphenol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) ) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants, such as polyethylene glycol (PEG).

In some embodiments, a buffer is included in the composition. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffers is used. The buffer or mixture thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st Edition (May 1, 2005).

The formulation of the binding molecule may include lyophilized formulations and aqueous solutions. The formulation or composition may also contain more than one active ingredient suitable for a specific indication, disease or condition that can be treated with the binding molecule or cell, preferably with complementary activities with the binding molecule or cell, wherein the individual activities do not adversely affect each other. Such active ingredients are suitably present in combination in an amount effective to achieve the intended purpose. Therefore, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active agents or drugs, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the binding molecule is administered in the form of a salt, for example, a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, metaphosphoric acid, nitric acid and sulfuric acid, and organic acids such as tartaric acid, acetic acid, citric acid, malic acid, lactic acid, fumaric acid, benzoic acid, glycolic acid, gluconic acid, succinic acid and arylsulfonic acids, such as p-toluenesulfonic acid.

The active ingredient can be entrapped in microcapsules, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. In certain embodiments, the pharmaceutical composition is formulated as an inclusion complex, such as a cyclodextrin inclusion complex; or as a liposome. Liposomes can be used to target host cells (e.g., T cells or NK cells) to specific tissues. There are a variety of methods available for preparing liposomes, such as those described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and U.S. Patents 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

In some embodiments, the pharmaceutical composition may be delivered using a timed release, delayed release, or sustained release delivery system such that delivery of the composition occurs prior to sensitization of the site to be treated and for a time sufficient to cause sensitization of the site to be treated. Various types of release delivery systems are available and known. Such systems may avoid repeated administration of the composition, thereby increasing convenience for individuals and physicians.

In some embodiments, the pharmaceutical composition contains an amount of binding molecules and/or cells that is effective for treating or preventing a disease or condition, such as a therapeutically effective amount or a prophylactically effective amount. In some embodiments, the therapeutic or prophylactic efficacy is monitored by periodic assessment of the treated individual. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until the desired suppression of disease symptoms occurs. However, other dosing regimens may be applicable and may be determined. The desired dose may be delivered by a single rapid administration of the composition, by multiple rapid administrations of the composition, or by continuous infusion of the composition.

In certain embodiments, in the case of genetically engineered cells containing a binding molecule, cells ranging from about 1 million to about 200 billion, such as about 1 million to about 50 billion (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 10 billion cells, about 1 ... or about 20 billion cells, or about 30 billion cells, or about 40 billion cells, or a range defined by any two of the foregoing values), or such as from about 10 million to about 100 billion cells (e.g., from about 20 million cells, from about 30 million cells, from about 40 million cells, from about 60 million cells, from about 70 million cells, from about 80 million cells, from about 10 million cells, from about 15 million cells, from about 20 million cells, from about 30 million cells, from about 40 million cells, from about 60 million cells, from about 70 million cells, from about 15 million cells, from about 10 million cells, from about 15 million cells, from about 20 million cells, from about 30 million cells, from about 40 million cells, from about 60 million cells, from about 70 million cells, from about 15 million cells, from about 15 million cells, from about 15 million cells, from about 20 million cells, from about 30 million cells, from about 40 million cells, from about 60 million cells, from about 70 million cells, from about cells, at or about 80 million cells, at or about 90 million cells, at or about 10 billion cells, at or about 25 billion cells, at or about 50 billion cells, at or about 75 billion cells, at or about 90 billion cells, or a range bounded by any two of the foregoing values), and in some cases, from or about 100 million cells to or about 50 billion cells (e.g., from or about 100 million cells to or about 50 billion cells). 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges, and/or such number of cells per kilogram of individual body weight.

The cells or binding molecules can be administered using standard administration techniques, formulations, and/or devices. Formulations and devices for storing and administering the compositions, such as syringes and vials, are provided. Administration of cells can be autologous or allogeneic. For example, immunoreactive cells or progenitor cells can be obtained from one individual and administered to the same individual or to a different compatible individual. Immunoreactive cells or their progeny derived from peripheral blood (e.g., from an in vivo, ex vivo, or ex vivo source) can be administered by local injection (including catheter administration), systemic injection, local injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing genetically modified immunoreactive cells), it is typically formulated into a unit dose injectable form (solution, suspension, emulsion).

Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, intracranial, intrathoracic, and intraperitoneal administration. In some embodiments, the cell population is administered to a subject by intravenous, intraperitoneal, or subcutaneous injection using peripheral systemic delivery.

In some embodiments, the composition is provided in the form of a sterile liquid formulation, such as an isotonic aqueous solution, suspension, emulsion, dispersion or viscous composition, which in some aspects can be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, administration of liquid compositions is more convenient to some extent, especially by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to allow a longer period of contact with a specific tissue. The liquid or viscous composition may include a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the binding molecule into a solvent, such as by mixing with a suitable carrier, diluent or excipient (such as sterile water, physiological saline, glucose, dextrose or the like).

Formulations for intravenous administration are generally sterile. Sterility can be readily achieved, for example, by filtration through a sterile filter membrane.

B. Therapeutic and preventive methods and uses

Also provided are methods of administering and uses of binding molecules, including TCRs and antigen-binding fragments thereof and antibodies or antigen-binding fragments thereof, and/or engineered cells expressing the binding molecules, such as for therapeutic and preventive uses. Such methods and uses include therapeutic methods and uses, for example, involving administering molecules, cells, or compositions containing the same to an individual suffering from a disease, condition, or disorder that expresses or is associated with HPV (e.g., HPV16), and/or wherein the cells or tissues express, for example, specifically express HPV16, such as HPV16 E6 or E7. In some embodiments, the molecules, cells, and/or compositions are administered in an effective amount to achieve treatment of the disease or disorder. Uses include uses of binding molecules and cells in such methods and treatments and in the preparation of medicaments for carrying out such therapeutic methods. In some embodiments, the methods are performed by administering a binding molecule or cell or a composition comprising the same to an individual who has, has had, or is suspected of having a disease or condition. In some embodiments, the methods thereby treat the disease, condition, or disorder in the individual.

As used herein, "treatment" (and its grammatical variations, such as "treat" or "treating") refers to the complete or partial improvement or reduction of a disease or condition or disorder, or symptoms, adverse effects or consequences, or phenotypes associated therewith. The desired therapeutic effects include (but are not limited to) preventing the occurrence or recurrence of the disease, relieving symptoms, reducing any direct or indirect pathological consequences of the disease, preventing cancer metastasis, slowing the rate of disease progression, improving or relieving the disease condition, and relieving or improving prognosis. These terms do not imply a complete cure of the disease or complete elimination of any symptoms or an effect on all symptoms or consequences.

As used herein, "delaying disease progression" means delaying, impeding, slowing, arresting, stabilizing, inhibiting and/or delaying the development of a disease (such as cancer). This delay may be of varying lengths of time, depending on the disease being treated and/or the individual's medical history. As will be apparent to one skilled in the art, a sufficient or significant delay may actually encompass prevention, such that the individual does not develop the disease. For example, advanced cancers, such as metastatic disease, may be delayed in their development.

As used herein, "prevention" includes providing a preventive effect on the appearance or recurrence of a disease in an individual who may be susceptible to the disease but has not yet been diagnosed with the disease. In some embodiments, the molecules and compositions provided are used to delay the development of a disease or slow the progression of a disease.

As used herein, "inhibiting" a function or activity is a decrease in function or activity when compared to otherwise identical conditions except for the condition or parameter of interest or compared to another condition. For example, a binding molecule or composition or cell that inhibits tumor growth reduces the tumor growth rate compared to the tumor growth rate in the absence of the binding molecule or composition or cell.

For example, an "effective amount" of a drug formulation, a binding molecule or cell or a combination of drugs, in the case of administration, refers to the amount that is effective in achieving the desired result at the necessary dose/amount and time period, such as a therapeutic or preventive result.

A "therapeutically effective amount" of an agent, such as a pharmaceutical formulation, binding molecule, or cell, is an amount effective to achieve the desired therapeutic outcome, such as for the treatment of a disease, condition, or disorder, and/or the pharmacokinetic or pharmacodynamic effect of the treatment, at the necessary dosage and duration. The therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the cell population being administered. In some embodiments, the methods provided involve administering an effective amount, such as a therapeutically effective amount of a binding molecule, cell, and/or composition.

"Prophylactically effective amount" means an amount that is effective in achieving the desired preventive result at the necessary dosage and duration. Usually, but not necessarily, because the preventive dose is used in an individual before or in the early stages of the disease, the preventive effective dose will be lower than the therapeutic effective dose.

As used herein, an "individual" is a mammal, such as a human or other animal, and typically a human.

The disease to be treated is cancer. In some embodiments, the cancer is an HPV-related cancer, and any HPV-related, such as HPV 16-related disease or condition, or a disease or condition that expresses an HPV oncoprotein, such as E6 or E7, such as an HPV 16 oncoprotein, such as HPV 16 E6 or E7. In certain diseases and conditions, viral proteins such as oncoproteins (such as HPV 16 E6 or E7) are expressed in or by malignant cells and cancers, and/or their peptide antigenic determinants are expressed on such malignant cancers or tissues, such as by MHC presentation. In some embodiments, the disease or condition is a cancer that expresses HPV16. In some embodiments, the cancer is a carcinoma, a melanoma, or other precancerous or cancerous condition caused by or otherwise associated with HPV, such as HPV-16. In some embodiments, the carcinoma may be a squamous cell or adenocarcinoma. In some embodiments, the disease or condition may be characterized by epithelial cell abnormalities associated with oncogenic HPV infection, such as koilocytosis; hyperkeratosis; precancerous conditions, including intraepithelial neoplasia or intraepithelial lesions; high-grade dysplasia; and invasive or malignant cancer. HPV 16-related diseases or conditions that can be treated include, but are not limited to, cervical cancer, uterine cancer, anal cancer, colorectal cancer, vaginal cancer, vulvar cancer, penile cancer, oropharyngeal cancer, tonsil cancer, pharyngeal cancer (pharynx cancer), laryngeal cancer (larynx cancer), oral cancer, skin cancer, esophageal cancer, head and neck cancer (such as squamous cell carcinoma (SCC) head and neck cancer) or small cell lung cancer. In some embodiments, the disease or condition is cervical cancer.

In some embodiments, the method may include steps or features for identifying individuals who have, are suspected of having, or are at risk of developing an HPV 16-related disease or condition (see, e.g., U.S. Patent Nos. 6,355,424 and 8,968,995) and/or the individual to be treated may be an individual identified as having such an HPV-related disease or condition or cancer, or as being at risk of developing or developing the disease. Thus, in some aspects, methods are provided for identifying individuals with diseases or conditions associated with HPV 16 E6 or E7 expression and selecting such individuals for treatment and/or treating such individuals, such as with provided HPV 16 binding molecules, including in some aspects selectively treating such individuals with cells engineered to express such binding molecules, including in some aspects any of an HPV 16 E6 or E7 TCR or antigen-binding fragment thereof or an anti-HPV 16 E6 or E7 antibody, such as antibody fragments and proteins containing the same, such as chimeric receptors, such as TCR-like CARs, and/or engineered cells expressing TCRs or CARs.

For example, an individual may be screened for a disease or condition associated with expression of HPV 16 E6 or E7, such as the presence of a cancer expressing HPV 16 E6 or E7. In some embodiments, the method comprises screening or detecting a disease associated with HPV 16 E6 or E7, such as the presence of a tumor. Thus, in some aspects, a sample may be obtained from a patient suspected of having a disease or condition associated with expression of HPV 16 E6 or E7 and analyzed for the amount of HPV 16 E6 or E7 expressed. In some aspects, an individual who tests positive for a disease or condition associated with HPV 16 E6 or E7 may be selected for treatment with the methods of the invention, and a therapeutically effective amount of a binding molecule described herein, a CAR expressing such a binding molecule, a cell containing a binding molecule, or a pharmaceutical composition thereof as described herein may be administered thereto. In some embodiments, the methods can be used to monitor the size or density of tissues (e.g., tumors) expressing HPV 16 E6 or E7 over time, such as before, during, or after treatment with the methods. In some aspects, individuals treated by the methods provided herein have been selected according to such methods or tested positive for HPV expression, such as before initiating treatment or during treatment.

In some embodiments, the provided HPV 16 binding molecules, including any of the HPV 16 E6 or E7 TCRs or antigen-binding fragments thereof or anti-HPV 16 E6 or E7 antibodies, such as antibody fragments and proteins containing them, such as chimeric receptors, such as TCR-like CARs, and/or engineered cells expressing TCRs or CARs, can be administered in combination with another therapeutic agent for treating HPV disease. For example, additional therapeutic treatment may include treatment with another anticancer agent for treating cervical cancer. The appropriate dosage of such co-administered agents can be reduced due to the combined action (synergistic effect) of the agents with the provided HPV 16 binding molecules.

In some embodiments, the individual has persistent or recurrent disease, for example after treatment with another HPV 16-specific binding molecule and/or cells expressing a binding molecule targeted to HPV 16 and/or other therapies, including chemotherapy, radiation and/or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT. In some embodiments, the administration is effective in treating the individual despite the individual having become resistant to another HPV 16-targeted therapy. In some embodiments, the individual has not relapsed, but is determined to be at risk for relapse, such as a high risk of relapse, and therefore the compound or composition is administered prophylactically, for example to reduce the likelihood of relapse or to prevent relapse.

In some embodiments, the treatment does not induce an immune response in the individual to the treatment and/or does not induce such a response to an extent that would interfere with effective treatment of the disease or condition. In some aspects, the level of immunogenicity and/or graft-versus-host response is less than that observed using a different but similar treatment. For example, in the case of a cell-transfer therapy, using cells expressing a TCR or CAR that includes a provided binding molecule, in some embodiments, the level of immunogenicity is reduced compared to a TCR or CAR that includes a different binding molecule.

In some embodiments, the method comprises administering cell therapy, wherein genetically engineered cells expressing a provided binding molecule are administered to an individual. Such administration can promote activation of cells (e.g., T cell activation) in an HPV16-targeted manner, resulting in destruction of cells targeted for the disease or disorder.

Thus, provided methods and uses include methods and uses for cell therapy. In some embodiments, the methods include administering cells or compositions containing cells to an individual, tissue, or cell, such as an individual, tissue, or cell suffering from, at risk for, or suspected of suffering from a disease, condition, or disorder. In some embodiments, cells, populations, and compositions are administered to an individual suffering from a specific disease or condition to be treated, for example, via cell therapy, such as T-cell therapy. In some embodiments, cells or compositions are administered to an individual, such as an individual suffering from a disease or condition or at risk for a disease or condition. In some aspects, the methods thereby treat, e.g., ameliorate one or more symptoms of a disease or condition, such as by reducing tumor burden in a cancer expressing HPV 16 E6 or E7.

Methods for administering cells for donor-receiver cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, donor-receiver T cell therapy is described in, for example, U.S. Patent Application Publication No. 2003/0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10): 577-85. See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.

In some embodiments, cell therapy, such as donor-transfer cell therapy, such as donor-transfer T cell therapy, is performed by autologous transfer, wherein cells are isolated and/or otherwise prepared from an individual to be treated with cell therapy or from a sample derived from such an individual. Thus, in some aspects, the cells are derived from an individual in need of treatment, such as a patient, and the cells are administered to the same individual after isolation and treatment.

In some embodiments, cell therapy, such as donor-receiver cell therapy, such as donor-receiver T cell therapy, is performed by allogeneic transfer, wherein cells are isolated and/or otherwise prepared from an individual (e.g., a first individual) other than the individual who will or ultimately receive the cell therapy. In such embodiments, the cells are then administered to a different individual (e.g., a second individual) of the same species. In some embodiments, the first and second individuals are genetically identical. In some embodiments, the first and second individuals are genetically similar. In some embodiments, the second individual expresses the same HLA class or supertype as the first individual.

In some embodiments, the subject to which the cell, cell population, or composition is administered is a primate, such as a human. In some embodiments, the primate is a monkey or ape. The subject can be male or female and can be any age-appropriate subject, including infants, young children, young adults, adults, and elderly subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent. In some embodiments, the patient or subject is a validated animal model for disease, administration of cell therapy, and/or for assessing toxicity outcomes, such as cytokine release syndrome (CRS).

The provided binding molecules, such as TCRs and antigen-binding fragments thereof and antibodies and antigen-binding fragments thereof, and cells expressing the same, can be administered by any suitable method, for example, by injection, such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, periocular injection or posterior juxta-scleral delivery. In some embodiments, they are administered parenterally, intrapulmonaryly and intranasally, and when local treatment is desired, by intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intracranial, intrathoracic, or subcutaneous administration. Dosing and administration may depend in part on whether the administration is brief or chronic. Various dosing schedules include, but are not limited to, single administration or multiple administrations at various time points, rapid administration, and pulse infusion.

For the prevention or treatment of a disease, the appropriate dose of the binding molecule or cell may depend on the type of disease to be treated, the type of binding molecule, the severity and course of the disease, whether the binding molecule is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the binding molecule, and the judgment of the attending physician. In some embodiments, the compositions and molecules and cells are suitable for administration to the patient at one time or over a series of treatments.

In certain embodiments, in the case of genetically engineered cells containing a binding molecule, cells ranging from about 1 million to about 200 billion, such as about 1 million to about 50 billion (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 1 billion cells, about 20 billion cells, about 10 billion cells, about 15 ... 20 billion cells or about 20 billion cells, 30 billion cells or about 30 billion cells, 40 billion cells or about 40 billion cells, or a range defined by any two of the foregoing values), or such as 10 million to 100 billion cells or about 100 billion cells (e.g., 20 million cells or about 20 million cells, 30 million cells or about 30 million cells, 40 million cells or about 40 million cells, 60 million cells or about 60 million cells, 70 million cells or about 70 million cells). 10,000,000 cells, 80,000,000 cells or about 80,000,000 cells, 90,000,000 cells or about 10 billion cells, 25,000,000,000 cells or about 50 billion cells, 75,000,000,000 cells or about 90 billion cells, or a range bounded by any two of the foregoing values), and in some cases, 100,000,000 cells or about 100,000,000 cells to 50,000,000,000 cells (e.g., 10,000,000,000 cells or about 100,000,000 cells). about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges, and/or such number of cells per kilogram of individual body weight. In some embodiments, in the case of genetically engineered cells comprising a binding molecule, 10 million cells or about 100 million cells, 1 billion cells or about 10 billion cells, 10 billion cells or about 10 billion cells, 100 billion cells or about 100 billion cells, or any value in between these ranges and/or per kilogram of body weight are administered to the individual. In addition, the dosage may vary depending on the disease or condition and/or patient and/or other treatment-specific properties.

Dosages may vary depending on the disease or condition and/or patient and/or other treatment-specific properties. In some embodiments, such values refer to the number of recombinant receptor-expressing cells.

In some embodiments, for example, where the individual is a human, the dose includes a total number of less than about 2×10 ¹¹ recombinant receptor (e.g., TCR)-expressing cells, for example, a total number of such cells in the range of between or about 1×10 ⁶ to or about 1.5×10 ¹¹ , such as a total number of 1×10 ⁷ , 3×10 ⁷ , 1×10 ⁸ , 5×10 ⁸ , 1×10 ⁹ , 1×10 ¹⁰ , 5×10 ¹⁰ , 1×10 ¹¹ , 1.25×10 ¹¹ such cells, or a range between any two of the foregoing values. In some embodiments, for example, where the individual is a human, the dose includes a total number of more than or about 1×10 ⁷ recombinant receptor (e.g., TCR)-expressing cells and a total number of less than or about 1×10 ¹¹ recombinant receptor (e.g., TCR)-expressing cells, such as between or about 1×10 ⁷ and or about 1×10 ¹¹ such cells, such as a total number of or about 5×10 ⁷ , 1×10 ⁸ , 5×10 ⁸ , 1×10 ⁹ , 1×10 ¹⁰ , 5×10 ¹⁰ , 7.5×10 ¹⁰ , 1×10 ¹¹ such cells, or a range between any two of the foregoing values.

In some embodiments, the dose of the genetically engineered cells comprises at or about 1×10 ⁶ to at or about 2×10 ¹¹ total TCR expressing cells, at or about 1×10 ⁶ to at or about 1.5×10 ¹¹ total TCR expressing cells, at or about 1×10 ⁶ to at or about 1×10 ¹¹ total TCR expressing cells, at or about 1×10 ⁶ to at or about 5×10 ¹⁰ total TCR expressing cells, at or about 1×10 ⁶ to at or about 1×10 ¹⁰ total TCR expressing cells, at or about 1×10 ⁶ to at or about 1×10 ⁹ total TCR expressing cells, at or about 1×10 ⁶ to at or about 5×10 10 total TCR expressing cells. ⁸ total TCR expressing cells, 1×10 ⁶ to 1×10 ⁸ total TCR expressing cells, 1×10 ⁶ to 5×10 ⁷ total TCR expressing cells, 1×10 ⁶ to 1×10 ⁷ total TCR expressing cells, 1×10 ⁶ to 5×10 ⁶ total TCR expressing cells, 1×10 ⁶ to 2.5×10 ⁶ total TCR expressing cells, 1×10 ⁶ to 2×10 ⁶ total TCR expressing cells, 2×10 ⁶ to 2×10 ¹¹ total TCR expressing cells, 2.5×10 ⁶ to 2×10 ¹¹ total TCR expressing cells, or about 5×10 ⁶ to 2×10 ¹¹ total TCR expressing cells, or about 1×10 ⁷ to 2×10 ¹¹ total TCR expressing cells, or about 3×10 ⁷ to 2×10 ¹¹ total TCR expressing cells, or about 1×10 ⁸ to 2×10 ¹¹ total TCR expressing cells, or about 5×10 ⁸ to 2×10 ¹¹ total TCR expressing cells, or about 1×10 ⁹ to 2×10 ¹¹ total TCR expressing cells, or about 1×10 ¹⁰ to 2×10 ¹¹ total TCR expressing cells, 5×10 ¹⁰ to 2×10 ¹¹ total TCR expressing cells, 1×10 ⁶ to 2×10 ¹¹ total TCR expressing cells, 1.5×10 ¹⁰ to 2×10 ¹¹ total TCR expressing cells. In some embodiments, the dose of genetically engineered cells comprises 1×10 ⁷ to 1×10 ¹¹ total TCR expressing cells, such as 1×10 ⁹ to 1×10 ¹⁰ total TCR expressing cells.

In some embodiments, the dose of genetically engineered cells comprises at least or about 2×10 ¹¹ TCR expressing cells, at least or about 1.75×10 ¹¹ TCR expressing cells, at least or about 1.5×10 ¹¹ TCR expressing cells, at least or about 1.25×10 ¹¹ TCR expressing cells, at least or about 1×10 ¹¹ TCR expressing cells, at least or about 7.5×10 ¹⁰ TCR expressing cells, at least or about 5×10 ¹⁰ TCR expressing cells, at least or about 2.5×10 ¹⁰ TCR expressing cells, at least or about 1×10 ¹⁰ TCR expressing cells, at least or about 5×10 ⁹ TCR expressing cells, at least or about 1×10 ⁹ TCR expressing cells, at least or about 5×10 ⁸ TCR expressing cells, at least or about 6×10 ⁷ TCR expressing cells, at least or about 3×10 ⁷ TCR expressing cells, at least or about 1×10 ⁷ TCR expressing cells, at least or about 5×10 ⁶ TCR expressing cells, at least or about 1×10 ⁶ TCR expressing cells.

In some embodiments, the dose of genetically engineered cells comprises 2×10 ¹¹ TCR expressing cells or about 1.75×10 ¹¹ TCR expressing cells, 1.5×10 11 TCR expressing cells or about 1.25×10 ¹¹ TCR expressing cells, 1×10 ¹¹ TCR expressing cells or about 7.5×10 ¹⁰ TCR expressing cells, 5×10 ¹⁰ TCR expressing cells or about 2.5×10 ¹⁰ TCR expressing cells, 1×10 ¹⁰ TCR expressing cells or about 1×10 ¹⁰ TCR expressing cells, 5×10 ⁹ TCR expressing cells or about 1×10 ⁹ TCR-expressing cells, is or is approximately 5×10 ⁸ TCR-expressing cells, is or is approximately 1×10 ⁸ TCR-expressing cells, is or is approximately 5×10 ⁷ TCR-expressing cells, is or is approximately 3×10 ⁷ TCR-expressing cells, is or is approximately 1×10 ⁷ TCR-expressing cells, is or is approximately 5×10 ⁶ TCR-expressing cells, is or is approximately 1×10 ⁶ TCR-expressing cells.

In some embodiments, a dose of cells, such as T cells expressing a recombinant receptor (e.g., TCR expressing cells), is administered to an individual as a single dose or only once over a period of two weeks, one month, three months, six months, one year, or longer. In some embodiments, one or more doses are administered to an individual.

In some embodiments, the binding molecule or cell is administered as part of a combination therapy, such as concurrently or sequentially in any order with another therapeutic intervention, such as another TCR, antibody or engineered cell or receptor or agent, such as a cytotoxic agent or therapeutic agent.

In some embodiments, cells or antibodies are co-administered with one or more other therapeutic agents or administered in combination with another therapeutic intervention simultaneously or sequentially in any order. In some cases, cells are co-administered with another therapy close enough in time that the cell population enhances the effect of the one or more other therapeutic agents or vice versa. In some embodiments, cells or antibodies are administered before one or more other therapeutic agents. In some embodiments, cells or antibodies are administered after one or more other therapeutic agents.

Once the cells are administered to a mammal (e.g., a human), in some embodiments, the biological activity of the engineered cell population and/or the binding molecule is measured by any of a variety of known methods. Assessment parameters include specific binding of engineered or natural T cells or other immune cells to antigens in vivo, for example, by imaging; or in vitro, for example, by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009); and Herman et al., J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of cells can also be measured by analyzing the expression and/or secretion of certain interleukins, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects, the biological activity is measured by assessing clinical outcomes, such as reduction in tumor burden or burden.

In some embodiments, the engineered cells are modified in various ways to increase their therapeutic or preventive efficacy. For example, in some embodiments, an engineered TCR expressed by an engineered cell or a CAR expressing an antibody is directly or indirectly bound to a targeting moiety via a linker. The practice of conjugating a compound (e.g., a TCR or CAR) to a targeting moiety is known in the art. See, e.g., Wadwa et al., J. Drug Targeting 3: 1 1 1 (1995) and U.S. Patent 5,087,616.

C. Diagnosis and Detection Methods

Methods are also provided, which involve using the provided binding molecules, such as TCRs or antigen-binding fragments thereof and antibodies and antigen-binding fragments thereof, for detecting HPV 16, such as HPV 16 E6 or HPV 16 E7, for example, in diagnostic and/or prognostic methods associated with a disease or condition expressing HPV 16. In some embodiments, the methods include incubating a biological sample with the binding molecule and/or administering the binding molecule to an individual. In certain embodiments, the biological sample includes cells or tissues, such as tumors or cancer tissues. In certain binding molecules directed to a region or peptide antigenic determinant of HPV 16, such as HPV 16 E6 or E7, and detecting whether a complex is formed between the binding molecule and the peptide antigenic determinant. Such methods can be in vitro or in vivo methods. In one embodiment, an anti-HPV 16 binding molecule is used to select individuals suitable for treatment with an anti-HPV 16 binding molecule or an engineered cell comprising such a molecule, for example where HPV 16, such as HPV 16 E6 or E7, is a biomarker for selecting patients.

In some embodiments, a sample, such as a cell, tissue sample, lysate, composition, or other sample derived therefrom, is contacted with a binding molecule and the binding or complex formation between the binding molecule and the sample (e.g., a region or antigenic determinant of HPV16 in the sample) is determined or detected. When binding is exhibited or detected in the test sample compared to a reference cell of the same tissue type, it can indicate the presence of a related disease or condition. In some embodiments, the sample is from human tissue.

Various methods known in the art for detecting specific binding molecule-antigen binding may be used. Exemplary immunoassays include fluorescence polarization immunoassay (FPIA), fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA). Indicator moieties or labeling groups may be attached to the binding molecules of the present invention and are selected to meet the requirements of the various uses of the method, which requirements are generally determined by the availability of analytical equipment and compatible immunoassay procedures. Exemplary labels include radionuclides (e.g., ¹²⁵ I, ¹³¹ I, ³⁵ S, ³ H, or ³² P), enzymes (e.g., alkaline phosphatase, horseradish peroxidase, luciferase, or β-galactosidase), fluorescent moieties or proteins (e.g., luciferin, rhodamine, phycoerythrin, GFP, or BFP), or luminescent moieties (e.g., Qdot ^™ nanoparticles supplied by Quantum Dot Corporation, Palo Alto, Calif.). General techniques for performing the various immunoassays mentioned above are known to those of ordinary skill in the art.

For diagnostic purposes, the binding molecule may be labeled with a detectable moiety, including but not limited to radioisotopes, fluorescent labels, and various enzyme-substrate labels known in the art. Methods for conjugating labels to binding molecules (e.g., TCRs or antibodies) are known in the art. In some embodiments, the binding molecule need not be labeled, and its presence can be detected using a labeled antibody that binds to the binding molecule.

In some embodiments, the provided binding molecules can be used in any known analytical method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Binding molecules can also be used in in vivo diagnostic assays, such as in vivo imaging. Typically, binding molecules are labeled with radionuclides (such as ¹¹¹ In, ⁹⁹ Tc, ¹⁴ C, ¹³¹ I, ¹²⁵ I, or ³ H) so that after administration to an individual, cells or tissues of interest can be localized in vivo. Binding molecules can also be used as staining reagents in pathology, for example using known techniques.

VI. Products

Also provided are articles of manufacture containing the provided binding molecules, such as TCRs, antibodies and CARs and/or engineered cells and/or compositions. The article of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials, such as glass or plastic. In some embodiments, the container contains a composition alone or in combination with another composition that is effective for treating, preventing and/or diagnosing a condition. In some embodiments, the container has a sterile access port. Exemplary containers include intravenous solution bags, vials, including those containers with stoppers that can be pierced by an injection needle. The label or package insert may indicate that the composition is used to treat a disease or condition manifesting or associated with HPV 16 E6 or E7. The article of manufacture may include (a) a first container containing a composition therein, wherein the composition includes an antibody or an engineered antigen receptor; and (b) a second container containing a composition therein, wherein the composition includes another agent, such as a cytotoxic agent or another therapeutic agent. The article of manufacture may further include instructions indicating that the composition can be used to treat a specific condition. Alternatively or additionally, the article of manufacture may further include another or the same container containing a pharmaceutically acceptable buffer. It may further include other materials, such as other buffers, diluents, filters, needles and/or syringes.

VII. Definition

Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or nouns used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and/or ease of reference, and the inclusion of such definitions herein is not necessarily to be construed as representing a substantial difference from what is commonly understood in the art.

The terms "polypeptide" and "protein" are used interchangeably and refer to polymers of amino acid residues and are not limited to a minimum length. Polypeptides, including provided antibodies and antibody chains and other peptides, such as linkers, can include amino acid residues, including natural and/or non-natural amino acid residues. Such terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation and the like. In some aspects, a polypeptide may contain modifications relative to the native or natural sequence, as long as the protein maintains the desired activity. Such modifications may be intentional, such as induced by site-directed mutagenesis; or may be accidental, such as through mutations in the host producing the protein or errors caused by PCR amplification.

An "isolated" nucleic acid is a nucleic acid molecule that has been separated from components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain nucleic acid molecules, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

"Isolated nucleic acid encoding TCR or antibody" refers to one or more nucleic acid molecules encoding TCR α or β chain (or fragments thereof) or antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell.

The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and the progeny produced therefrom, regardless of the number of generations. Progeny may not be completely identical to the parent cell in terms of nucleic acid content, but may contain mutations. This includes screening or selecting for mutant progeny with the same function or biological activity as the original transformed cell.

As used herein, "percent amino acid sequence identity (%)" and "percent identity" when used with respect to an amino acid sequence (reference polypeptide sequence) are defined as the percentage of amino acid residues in a candidate sequence (e.g., an antibody or fragment of the present invention) that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the candidate sequence with the reference polypeptide sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

Amino acid substitutions may include replacement of one amino acid with another in a polypeptide. Amino acid substitutions may be introduced into binding molecules of interest (e.g., TCRs or antibodies) and the products screened for desired activities, such as retained/improved antigen binding, reduced immunogenicity, or improved cytolytic activity.

Amino acids can generally be grouped according to the following common side chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

In some embodiments, conservative substitutions may involve exchanging a member of one of these classes with another member of the same class. In some embodiments, non-conservative amino acid substitutions may involve exchanging a member of one of these classes with another class.

As used herein, the term "vector" refers to a nucleic acid molecule that is capable of transmitting another nucleic acid molecule to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

The term "drug package insert" is used to refer to instructions customarily included in the commercial packaging of therapeutic products that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

Unless the context clearly indicates otherwise, the singular forms "a/an" and "the" as used herein include plural referents. For example, "a/an" means "at least one" or "one or more". It should be understood that the aspects and variations described herein include "consisting of" and/or "consisting primarily of" aspects and variations.

Throughout the present invention, various aspects of the claimed subject matter are presented in the form of ranges. It should be understood that the description in range form is for convenience and brevity only and should not be interpreted as an inflexible limitation on the scope of the claimed subject matter. Therefore, the description of the range should be regarded as having specifically disclosed all possible sub-ranges and individual numerical values within the range. For example, where a range of values is provided, it should be understood that each intermediate value between the upper and lower limits of the range and any other stated or intermediate values within the stated range are covered by the claimed subject matter. The upper and lower limits of such smaller ranges may be independently included in the smaller range and also covered by the claimed subject matter, subject to any specific exclusive limitations within the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

As used herein, the term "about" refers to the common error range of the corresponding value that is readily known to a person skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments for the value or parameter itself. For example, a description referring to "about X" includes a description of "X".

As used herein, a composition refers to any mixture of two or more products, substances or compounds (including cells). It can be a solution, a suspension, a liquid, a powder, a paste, an aqueous solution, a non-aqueous solution or any combination thereof.

As used herein, the statement that a cell or cell population is "positive" for a particular marker means that the presence of the particular marker, typically a surface marker, can be detected on or in the cell. When referring to a surface marker, the term refers to the presence of surface expression, such as detected by flow cytometry, for example, staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is detectable by flow cytometry to an extent that is substantially greater than the extent of staining detected using isotype-matched controls performed under otherwise identical conditions, and/or substantially similar to the extent of staining of cells known to be positive for the marker, and/or substantially greater than the extent of staining of cells known to be negative for the marker.

As used herein, the statement that a cell or cell population is "negative" for a particular marker means that the presence of the particular marker, typically a surface marker, is substantially undetectable on or in the cell. In reference to a surface marker, the term refers to the absence of surface expression, as detected by flow cytometry, e.g., staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the staining is not detected by flow cytometry to an extent that is substantially greater than the extent of staining detected using isotype-matched controls performed under otherwise identical conditions and/or substantially less than the extent of staining of cells known to be positive for the marker and/or to an extent that is substantially similar to the extent of staining of cells known to be negative for the marker.

VIII. Exemplary Embodiments

The provided embodiments are:

1. A T cell receptor (TCR) or an antigen-binding fragment thereof, comprising an α chain containing a variable α (Vα) region and a β chain containing a variable β (Vβ) region, wherein: The Vα region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 47, 77, 103, 126, 149, 169, 195 or 221, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith; and/or the Vβ region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 60, 88, 114, 137, 157, 180, 206 or 232, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith.

2. A T cell receptor (TCR) or an antigen-binding fragment thereof, comprising an α chain containing a variable α (Vα) region and a β chain containing a variable β (Vβ) region, wherein: the Vα region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 47, 77, 103, 126, 149, 169, 195 or 221, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith; and the Vβ region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 60, 88, 114, 137, 157, 180, 206 or 232, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith.

3. A T cell receptor (TCR) or an antigen-binding fragment thereof according to Example 1 or Example 2, wherein: the Vα region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 47, 149, 169, 195 or 221, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith; or The Vα region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 103, 126, or an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith.

4. The T cell receptor (TCR) or antigen-binding fragment thereof of any one of embodiments 1 to 3, wherein: the Vβ region comprises the amino acid sequence shown in any one of SEQ ID NOs: 60, 180 or 232, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; the Vβ region comprises the amino acid sequence shown in SEQ ID NO: 206, or an amino acid sequence having at least 90.5%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; the Vβ region comprises the amino acid sequence shown in SEQ ID NO: 114 or 137, or an amino acid sequence having at least 97.5%, 98% or 99% sequence identity thereto; the Vβ region comprises the amino acid sequence shown in SEQ ID NO: 1 The amino acid sequence shown in SEQ ID NO: 157 or an amino acid sequence having at least 98.5% or 99% sequence identity therewith; or the Vβ region comprises the amino acid sequence shown in SEQ ID NO: 88 or an amino acid sequence having at least 99.5% sequence identity therewith.

5. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 4, wherein the TCR or antigen-binding fragment thereof binds to or recognizes a peptide antigenic determinant of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule, the peptide antigenic determinant being or comprising E7(11-19)YMLDLQPET (SEQ ID NO: 271).

6. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 5, wherein the TCR or antigen-binding fragment thereof, when expressed on the surface of a T cell, stimulates cytotoxic activity against a target cancer cell, wherein the target cancer cell contains an HPV DNA sequence or expresses HPV 16, and wherein the target cancer cell is SCC152.

7. A TCR or antigen binding fragment as in Example 7, wherein the target cancer cell expresses a peptide antigen determinant of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule, and the peptide antigen determinant is or comprises E7(11-19)YMLDLQPET (SEQ ID NO: 271).

8. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 7, wherein: the Vα region comprises a complementary determining region 1 (CDR-1), CDR-2 and CDR-3, which respectively comprise the amino acid sequences of the CDR-1, the CDR-2 and the CDR-3 contained in the Vα region amino acid sequence shown in any one of SEQ ID NOs: 47, 77, 103, 126, 149, 169, 195 or 221; and/or the Vβ region comprises a complementary determining region 1 (CDR-1), CDR-2 and CDR-3, which respectively comprise the amino acid sequences of the CDR-1, the CDR-2 and the CDR-3 contained in the Vβ region amino acid sequence shown in any one of SEQ ID NOs: 60, 88, 114, 137, 157, 180, 206 or 232.

9. A TCR or antigen-binding fragment according to any one of embodiments 1 to 8, wherein: the Vα region comprises a complementary determining region 3 (CDR-3) comprising an amino acid sequence as shown in any one of SEQ ID NOs: 50, 80, 106, 129, 150, 172, 198 or 224; and/or the Vβ region comprises a complementary determining region 3 (CDR-3) comprising an amino acid sequence as shown in any one of SEQ ID NOs: 63, 91, 115, 138, 158, 183, 209 or 235.

10. The TCR or antigen-binding fragment thereof according to Example 9, wherein the Vα region comprises: a complementary determining region 1 (CDR-1) comprising an amino acid sequence as shown in any one of SEQ ID NOs: 48, 78, 104, 127, 170, 196 or 222; and/or a complementary determining region 2 (CDR-2) comprising an amino acid sequence as shown in any one of SEQ ID NOs: 49, 79, 105, 128, 171, 197 or 223.

11. The TCR or antigen-binding fragment thereof of Example 9 or Example 10, wherein the Vβ region comprises: a complementary determining region 1 (CDR-1) comprising an amino acid sequence as shown in any one of SEQ ID NOs: 61, 89, 181, 207 or 233; and/or a complementary determining region 2 (CDR-2) comprising an amino acid sequence as shown in SEQ ID NOs: 62, 90, 182, 208 or 234.

12. The TCR or antigen-binding fragment thereof of any one of embodiments 1 to 11, wherein: the Vα region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 48, 49 and 50, respectively, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 61, 62 and 63, respectively; the Vα region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 78, 79 and 80, respectively, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 89, 90 and 91, respectively; the Vα region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: NO: 104, 105 and 106, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 89, 90 and 115; the Vα region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 127, 128 and 129, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 89, 90 and 138; the Vα region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 127, 128 and 150, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: NOs: 89, 90 and 158; the Vα region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 170, 171 and 172, respectively, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 181, 182 and 183, respectively; the Vα region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 196, 197 and 198, respectively, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 207, 208 and 209, respectively; or the Vα region comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: The amino acid sequences of SEQ ID NOs: 222, 223 and 224, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 233, 234 and 235.

13. The TCR or antigen-binding fragment thereof of any one of embodiments 1 to 10, wherein: the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 47 and 60, respectively, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 77 and 88, respectively, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 77 and 88, respectively, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99.5% sequence identity thereto; the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: ID NO: 103 and 114 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98% or 99% sequence identity therewith; the Vα region and the Vβ region respectively comprise the amino acid sequence of SEQ ID NO: 126 and 137 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97.5%, 98% or 99% sequence identity therewith; the Vα region and the Vβ region respectively comprise the amino acid sequence of SEQ ID NO: 149 and 157 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98.5% or 99% sequence identity therewith; the Vα region and the Vβ region respectively comprise the amino acid sequence of SEQ ID NO: NO: 169 and 180 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith; the Vα region and the Vβ region respectively comprise the amino acid sequence of SEQ ID NO: 195 and 206 or an amino acid sequence having at least 90.5%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith; or the Vα region and the Vβ region respectively comprise the amino acid sequence of SEQ ID NO: 221 and 232 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith.

14. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 12, wherein: the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 47 and 60, respectively; the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 77 and 88, respectively; the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 77 and 88, respectively; the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 103 and 114, respectively; the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 126 and 137, respectively; the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 149 and 157, respectively; the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 169 and 180, respectively; the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: The amino acid sequences of SEQ ID NOs: 195 and 206; or the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 221 and 232, respectively.

15. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 14, wherein: the Vα region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 48, 49 and 50; and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 61, 62 and 63; and/or the Vα region and the Vβ region respectively comprise the amino acid sequences of SEQ ID NOs: 47 and 60.

16. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 14, wherein: the Vα region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 196, 197 and 198; and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 207, 208 and 209; and/or the Vα region and the Vβ region respectively comprise the amino acid sequences of SEQ ID NOs: 195 and 206.

17. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 14, wherein: the Vα region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 222, 223 and 224; and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 233, 234 and 235; and/or the Vα region and the Vβ region respectively comprise the amino acid sequences of SEQ ID NOs: 221 and 232.

18. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 17, wherein the α chain further comprises an α constitutive (Cα) region and/or the β chain further comprises a β constitutive (Cβ) region.

19. The TCR or antigen-binding fragment thereof according to Example 18, wherein the Cα region and the Cβ region are mouse constant regions.

20. The TCR or antigen-binding fragment thereof according to Example 18, wherein the Cα region and the Cβ region are human constant regions.

21. A TCR or antigen-binding fragment thereof according to any one of embodiments 18 to 20, wherein the Cα region and/or the Cβ region comprises the introduction of one or more cysteine residues capable of forming one or more non-native disulfide bridges between the α chain and the β chain.

22. A TCR or antigen-binding fragment thereof according to any one of embodiments 18, 19 and 21, wherein: the Cα region comprises the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 90% sequence identity therewith.

23. The TCR or antigen-binding fragment thereof of any one of embodiments 18, 19, 21 and 22, wherein: a) the α chain comprises the amino acid sequence shown in SEQ ID NO: 44, 74, 100, 123, 146, 166, 192 or 218, or an amino acid sequence having at least 90% sequence identity thereto; and/or the β chain comprises the amino acid sequence shown in SEQ ID NO: 57, 85, 111, 134, 154, 177, 203 or 229, or an amino acid sequence having at least 90% sequence identity thereto; or b) the α chain and the β chain comprise the amino acid sequences of SEQ ID NO: 44 and 57, respectively; the α chain and the β chain comprise the amino acid sequences of SEQ ID NO: 74 and 85, respectively; the α chain and the β chain comprise the amino acid sequences of SEQ ID NO: 57, 85, 111, 134, 154, 177, 203 or 229, respectively. NO: 100 and 111; the α chain and the β chain respectively comprise the amino acid sequences of SEQ ID NO: 123 and 134; the α chain and the β chain respectively comprise the amino acid sequences of SEQ ID NO: 146 and 154; the α chain and the β chain respectively comprise the amino acid sequences of SEQ ID NO: 166 and 177; the α chain and the β chain respectively comprise the amino acid sequences of SEQ ID NO: 192 and 202; or the α chain and the β chain respectively comprise the amino acid sequences of SEQ ID NO: 218 and 229.

24. A TCR or antigen-binding fragment thereof according to any one of embodiments 18, 20 and 21, wherein: the Cα region comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 90% sequence identity therewith.

25. A TCR or antigen-binding fragment thereof according to any one of embodiments 18, 20 and 21, wherein: the Cα region comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 350 or an amino acid sequence having at least 90% sequence identity therewith.

26. A TCR or antigen-binding fragment thereof according to any one of embodiments 21 to 25, wherein the introduction of one or more cysteine residues comprises replacing a non-cysteine residue with a cysteine residue.

27. A TCR or antigen-binding fragment thereof according to any one of embodiments 21 to 26, wherein the Cα region comprises a cysteine at a position corresponding to position 48 as shown in any one of SEQ ID NOs: 333-337 or at a position corresponding to position 49 as shown in SEQ ID NOs: 338 or 341; and/or the Cβ region comprises a cysteine at a position corresponding to position 57 as shown in SEQ ID NOs: 339 or 340 or at a position corresponding to position 58 as shown in SEQ ID NOs: 342 or 343.

28. The TCR or antigen-binding fragment thereof of any one of embodiments 18, 20, 21 and 24 to 27, wherein: a) the α chain comprises the amino acid sequence shown in SEQ ID NO: 43, 73, 99, 122, 145, 165, 191 or 217, or an amino acid sequence having at least 90% sequence identity thereto; and/or the β chain comprises the amino acid sequence shown in SEQ ID NO: 56, 84, 110, 133, 153, 176, 202 or 228, or an amino acid sequence having at least 90% sequence identity thereto; or b) the α chain and the β chain comprise the amino acid sequences of SEQ ID NO: 43 and 56, respectively; the α chain and the β chain comprise the amino acid sequences of SEQ ID NO: 73 and 84, respectively; the α chain and the β chain comprise the amino acid sequences of SEQ ID NO: 56, 84, 110, 133, 153, 176, 202 or 228, respectively. NO: 99 and 110; the α chain and the β chain respectively comprise the amino acid sequences of SEQ ID NO: 122 and 133; the α chain and the β chain respectively comprise the amino acid sequences of SEQ ID NO: 145 and 153; the α chain and the β chain respectively comprise the amino acid sequences of SEQ ID NO: 165 and 176; the α chain and the β chain respectively comprise the amino acid sequences of SEQ ID NO: 191 and 202; or the α chain and the β chain respectively comprise the amino acid sequences of SEQ ID NO: 217 and 228.

29. A TCR or antigen-binding fragment thereof according to any one of embodiments 18, 20, 21, 23 and 25 to 27, wherein: a) the α chain comprises an amino acid sequence as shown in SEQ ID NO: 43, 73, 99, 122, 145, 165, 191 or 217 or an amino acid sequence having at least 90% sequence identity therewith; and/or the β chain comprises an amino acid sequence as shown in SEQ ID NO: 359, 370, 371, 372, 373, 374, 375 or 376 or an amino acid sequence having at least 90% sequence identity therewith; or b) the α chain and the β chain comprise the amino acid sequences of SEQ ID NO: 43 and 359, respectively; the α chain and the β chain comprise the amino acid sequences of SEQ ID NO: 73 and 370, respectively; the α chain and the β chain comprise the amino acid sequences of SEQ ID NO: NO: 99 and 371; the α chain and the β chain respectively comprise the amino acid sequences of SEQ ID NO: 122 and 372; the α chain and the β chain respectively comprise the amino acid sequences of SEQ ID NO: 145 and 373; the α chain and the β chain respectively comprise the amino acid sequences of SEQ ID NO: 165 and 374; the α chain and the β chain respectively comprise the amino acid sequences of SEQ ID NO: 191 and 375; or the α chain and the β chain respectively comprise the amino acid sequences of SEQ ID NO: 217 and 376.

30. The TCR or antigen-binding fragment thereof of any one of embodiments 1 to 29, wherein the TCR or antigen-binding fragment comprises: (i) the α chain and the β chain comprising the amino acid sequences of SEQ ID NOs: 44 and 57, respectively; (ii) the α chain and the β chain comprising the amino acid sequences shown in SEQ ID NOs: 43 and 56, respectively; or (iii) the α chain and the β chain independently exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with the α chain and the β chain sequences in (i), respectively, or with the α chain and the β chain sequences in (ii), respectively, wherein the α chain comprises CDR-1, CDR-2 and CDR-3, which respectively comprise SEQ ID NOs: The amino acid sequence of SEQ ID NO: 48, 49 and 50, and the β chain comprises CDR-1, CDR-2 and CDR-3, which comprises the amino acid sequence of SEQ ID NO: 61, 62 and 63.

31. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 30, wherein the TCR or antigen-binding fragment comprises an alpha chain and a beta chain independently exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with the alpha chain and the beta chain sequences comprising the amino acid sequences of SEQ ID NOs: 43 and 56, respectively, wherein the alpha chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 48, 49 and 50, respectively, and the beta chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 61, 62 and 63.

32. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 31, wherein the TCR or antigen-binding fragment comprises an α chain and a β chain comprising the amino acid sequences of SEQ ID NOs: 43 and 56, respectively.

33. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 31, wherein the TCR or antigen-binding fragment comprises an α chain and a β chain comprising the amino acid sequences of SEQ ID NO: 43 and 359, respectively.

34. The TCR or antigen-binding fragment thereof of any one of embodiments 1 to 29, wherein the TCR or antigen-binding fragment comprises: (i) the α chain and the β chain comprising the amino acid sequences of SEQ ID NOs: 192 and 203, respectively; (ii) the α chain and the β chain comprising the amino acid sequences shown in SEQ ID NOs: 191 and 202, respectively; or (iii) the α chain and the β chain independently exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with the α chain and the β chain sequences in (i), respectively, or with the α chain and the β chain sequences in (ii), respectively, wherein the α chain comprises CDR-1, CDR-2 and CDR-3, which respectively comprise SEQ ID NOs: The amino acid sequences of SEQ ID NOs: 196, 197 and 198, and the β chain comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 207, 208 and 209.

35. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 29 and 34, wherein the TCR or antigen-binding fragment comprises an α chain and a β chain containing the amino acid sequences shown in SEQ ID NO: 191 and SEQ ID NO: 375.

36. The TCR or antigen-binding fragment thereof of any one of embodiments 1 to 29, wherein the TCR or antigen-binding fragment comprises: (i) the α chain and the β chain comprising the amino acid sequences of SEQ ID NOs: 218 and 229, respectively; (ii) the α chain and the β chain comprising the amino acid sequences shown in SEQ ID NOs: 217 and 228, respectively; or (iii) the α chain and the β chain independently exhibiting at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with the α chain and the β chain sequences in (i), respectively, or with the α chain and the β chain sequences in (ii), respectively, wherein the α chain comprises CDR-1, CDR-2 and CDR-3, which respectively comprise SEQ ID NOs: The amino acid sequences of SEQ ID NOs: 222, 223 and 224, and the β chain comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 233, 234 and 235.

37. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 29 and 36, wherein the TCR or antigen-binding fragment comprises an α chain and a β chain comprising the amino acid sequences of SEQ ID NO: 217 and 376, respectively.

38. A T cell receptor (TCR) or an antigen-binding fragment thereof, comprising an α chain containing a variable α (Vα) region and a β chain containing a variable β (Vβ) region, wherein: the Vα region comprises an amino acid sequence as shown in any one of SEQ ID NO: 247 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and/or the Vβ region comprises an amino acid sequence as shown in any one of SEQ ID NO: 258 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

39. A T cell receptor (TCR) or an antigen-binding fragment thereof, comprising an α chain containing a variable α (Vα) region and a β chain containing a variable β (Vβ) region, wherein: the Vα region comprises an amino acid sequence as shown in any one of SEQ ID NO: 247 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and the Vβ region comprises an amino acid sequence as shown in any one of SEQ ID NO: 258 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

40. The TCR or antigen-binding fragment thereof of Example 38 or Example 39, wherein: the Vα region comprises an amino acid sequence as shown in any one of SEQ ID NO: 247 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith; and/or the Vβ region comprises an amino acid sequence as shown in any one of SEQ ID NO: 258 or an amino acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith.

41. A TCR or antigen-binding fragment thereof according to any one of embodiments 38 to 40, wherein the TCR or antigen-binding fragment thereof binds to or recognizes a peptide antigenic determinant of human papillomavirus (HPV) 16 E6 in the context of an MHC molecule, the peptide antigenic determinant being or comprising E6(29-38)TIHDIILECV (SEQ ID NO: 268).

42. The TCR or antigen-binding fragment of any one of embodiments 38 to 41, wherein: the Vα region comprises a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 250 or a CDR3 contained in the amino acid sequence shown in SEQ ID NO: 247; and/or the Vβ region comprises a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 261 or a CDR3 contained in the amino acid sequence shown in SEQ ID NO: 258.

43. The TCR or antigen-binding fragment of Example 42, wherein the Vα region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 248 or a CDR-1 contained in the amino acid sequence shown in SEQ ID NO: 247; and/or a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 249 or a CDR-2 contained in the amino acid sequence shown in SEQ ID NO: 247.

44. The TCR or antigen-binding fragment of Example 42 or Example 43, wherein the Vβ region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 259 or a CDR-1 contained in the amino acid sequence shown in SEQ ID NO: 258; and/or a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 260 or a CDR-2 contained in the amino acid sequence shown in SEQ ID NO: 258.

45. A TCR or antigen-binding fragment thereof according to any one of embodiments 38 to 44, wherein: the Vα region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 248; a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 249; and/or a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 250; and/or the Vβ region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 259; a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 260; and/or a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 261.

46. A TCR or antigen-binding fragment thereof according to any one of embodiments 38 to 45, wherein: the Vα region comprises a complementary determining region 1 (CDR-1), CDR-2 and CDR-3, which respectively comprise the CDR-1, CDR-2 and CDR-3 amino acid sequences contained in the Vα region amino acid sequence shown in SEQ ID NO: 247; and/or the Vβ region comprises a complementary determining region 1 (CDR-1), CDR-2 and CDR-3, which respectively comprise the CDR-1, CDR-2 and CDR-3 amino acid sequences contained in the Vβ region amino acid sequence shown in SEQ ID NO: 258.

47. A TCR or antigen-binding fragment thereof according to any one of embodiments 38 to 46, wherein: the Vα region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 248, 249 and 250, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 259, 260 and 261.

48. A TCR or antigen-binding fragment thereof according to any one of embodiments 38 to 47, wherein the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 247 and 258, respectively, or amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith.

49. A TCR or antigen-binding fragment thereof according to any one of embodiments 38 to 48, wherein: the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 247 and 258, respectively.

50. A TCR or antigen-binding fragment thereof according to any one of embodiments 38 to 49, wherein the α chain further comprises an α constitutive (Cα) region and/or the β chain further comprises a β constitutive (Cβ) region.

51. The TCR or antigen-binding fragment thereof of Example 50, wherein the Cα region and the Cβ region are mouse constant regions.

52. The TCR or antigen-binding fragment thereof according to Example 50, wherein the Cα region and the Cβ region are human constant regions.

53. A TCR or antigen-binding fragment thereof according to any one of embodiments 50 to 52, wherein the Cα region and/or the Cβ region comprises the introduction of one or more cysteine residues capable of forming one or more non-native disulfide bridges between the α chain and the β chain.

54. A TCR or antigen-binding fragment thereof according to any one of embodiments 50, 51 and 53, wherein: the Cα region comprises the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 90% sequence identity therewith.

55. The TCR or antigen-binding fragment thereof of any one of embodiments 50, 51, 53 and 54, wherein: a) the alpha chain comprises the amino acid sequence shown in SEQ ID NO: 244, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and/or the beta chain comprises the amino acid sequence shown in SEQ ID NO: 255, or an amino acid sequence having at least 90% sequence identity thereto, wherein the alpha chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 248, 249 and 250, respectively, and the beta chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 248, 249 and 250, respectively. NO: 259, 260 and 261; or b) the α chain and the β chain comprise the amino acid sequences of SEQ ID NO: 244 and 255, respectively.

56. A TCR or antigen-binding fragment thereof according to any one of embodiments 50, 52 and 53, wherein: The Cα region comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 90% sequence identity therewith.

57. A TCR or antigen-binding fragment thereof according to any one of embodiments 50, 52 and 53, wherein: the Cα region comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 350 or an amino acid sequence having at least 90% sequence identity therewith.

58. A TCR or antigen-binding fragment thereof according to any one of embodiments 53 to 57, wherein the introduction of one or more cysteine residues comprises replacing a non-cysteine residue with a cysteine residue.

59. A TCR or antigen-binding fragment thereof according to any one of embodiments 53 to 58, wherein the Cα region comprises cysteine at a position corresponding to position 48 as shown in any one of SEQ ID NOs: 333-337 or at a position corresponding to position 49 as shown in SEQ ID NOs: 338 or 341; and/or the Cβ region comprises cysteine at a position corresponding to position 57 as shown in SEQ ID NOs: 339 or 340 or at a position corresponding to position 58 as shown in SEQ ID NOs: 342 or 343.

60. The TCR or antigen-binding fragment thereof of any one of embodiments 38 to 59, wherein: a) the alpha chain comprises the amino acid sequence shown in SEQ ID NO: 243, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and/or the beta chain comprises the amino acid sequence shown in SEQ ID NO: 254, or an amino acid sequence having at least 90% sequence identity thereto, wherein the alpha chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 248, 249 and 250, respectively, and the beta chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 259, 260 and 261, respectively; or b) the alpha chain and the beta chain comprise the amino acid sequences of SEQ ID NOs: NO: Amino acid sequence of 243 and 254.

61. The TCR or antigen-binding fragment thereof of any one of embodiments 38 to 59, wherein: a) the alpha chain comprises the amino acid sequence shown in SEQ ID NO: 243, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and/or the beta chain comprises the amino acid sequence shown in SEQ ID NO: 377, or an amino acid sequence having at least 90% sequence identity thereto, optionally wherein the alpha chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 248, 249 and 250, respectively, and the beta chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 259, 260 and 261, respectively; or b) the alpha chain and the beta chain comprise the amino acid sequences of SEQ ID NOs: NO: Amino acid sequence of 243 and 377.

62. A T cell receptor (TCR) or an antigen binding fragment thereof, comprising an α chain containing a variable α (Vα) region and a β chain containing a variable β (Vβ) region, wherein: the Vα region comprises an amino acid sequence as shown in any one of SEQ ID NO: 9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and/or the Vβ region comprises an amino acid sequence as shown in any one of SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

63. A T cell receptor (TCR) or an antigen-binding fragment thereof, comprising an α chain containing a variable α (Vα) region and a β chain containing a variable β (Vβ) region, wherein: the Vα region comprises an amino acid sequence as set forth in any one of SEQ ID NO: 9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and the Vβ region comprises an amino acid sequence as set forth in any one of SEQ ID NO: 24 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

64. The TCR or antigen-binding fragment thereof of embodiment 62 or embodiment 63, wherein: the Vα region comprises an amino acid sequence as shown in any one of SEQ ID NO: 9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith; and/or the Vβ region comprises an amino acid sequence as shown in any one of SEQ ID NO: 24 or an amino acid sequence having at least 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith.

65. A TCR or antigen-binding fragment thereof according to any one of embodiments 62 to 64, wherein the TCR or antigen-binding fragment thereof, when expressed on the surface of a T cell, stimulates cytotoxic activity against a target cancer cell, optionally wherein the target cancer cell contains an HPV DNA sequence or expresses HPV 16 and/or optionally wherein the target cancer cell is SCC152.

66. A TCR or antigen-binding fragment according to any one of embodiments 62 to 65, wherein: the Vα region comprises a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 12 or a CDR3 contained in the amino acid sequence shown in SEQ ID NO: 9; and/or the Vβ region comprises a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 27 or a CDR3 contained in the amino acid sequence shown in SEQ ID NO: 24.

67. The TCR or antigen-binding fragment of Example 66, wherein the Vα region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 10 or a CDR-1 contained in the amino acid sequence shown in SEQ ID NO: 9; and/or a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 11 or a CDR-2 contained in the amino acid sequence shown in SEQ ID NO: 9.

68. The TCR or antigen-binding fragment of Example 66 or Example 67, wherein the Vβ region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 25 or a CDR-1 contained in the amino acid sequence shown in SEQ ID NO: 24; and/or a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 26 or a CDR-2 contained in the amino acid sequence shown in SEQ ID NO: 24.

69. A TCR or antigen-binding fragment thereof according to any one of embodiments 62 to 68, wherein: the Vα region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 10; a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 11; and/or a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 12; and/or the Vβ region comprises: a complementary determining region 1 (CDR-1) comprising the amino acid sequence shown in SEQ ID NO: 25; a complementary determining region 2 (CDR-2) comprising the amino acid sequence shown in SEQ ID NO: 26; and/or a complementary determining region 3 (CDR-3) comprising the amino acid sequence shown in SEQ ID NO: 27.

70. A TCR or antigen-binding fragment thereof according to any one of embodiments 62 to 69, wherein: The Vα region comprises complementary determining region 1 (CDR-1), CDR-2 and CDR-3, which respectively comprise the CDR-1, CDR-2 and CDR-3 amino acid sequences contained in the Vα region amino acid sequence shown in SEQ ID NO: 9; and/or the Vβ region comprises complementary determining region 1 (CDR-1), CDR-2 and CDR-3, which respectively comprise the CDR-1, CDR-2 and CDR-3 amino acid sequences contained in the Vβ region amino acid sequence shown in SEQ ID NO: 24.

71. A TCR or antigen-binding fragment thereof according to any one of embodiments 62 to 70, wherein: the Vα region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 10, 11 and 12, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 25, 26 and 27.

72. A TCR or antigen-binding fragment thereof according to any one of embodiments 62 to 71, wherein the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 9 and 24, respectively, or amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith.

73. A TCR or antigen-binding fragment thereof according to any one of embodiments 62 to 72, wherein the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 9 and 24, respectively.

74. A TCR or antigen-binding fragment thereof according to any one of embodiments 62 to 73, wherein the α chain further comprises an α constitutive (Cα) region and/or the β chain further comprises a β constitutive (Cβ) region.

75. The TCR or antigen-binding fragment thereof according to Example 74, wherein the Cα region and the Cβ region are mouse constant regions.

76. The TCR or antigen-binding fragment thereof according to Example 74, wherein the Cα region and the Cβ region are human constant regions.

77. A TCR or antigen-binding fragment thereof according to any one of embodiments 74 to 76, wherein the Cα region and/or the Cβ region comprises the introduction of one or more cysteine residues capable of forming one or more non-native disulfide bridges between the α chain and the β chain.

78. A TCR or antigen-binding fragment thereof according to any one of embodiments 74, 75 and 77, wherein: the Cα region comprises the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 30 or an amino acid sequence having at least 90% sequence identity therewith.

79. The TCR or antigen-binding fragment thereof of any one of embodiments 74, 75, 77 and 78, wherein: a) the alpha chain comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and/or the beta chain comprises the amino acid sequence shown in SEQ ID NO: 21, or an amino acid sequence having at least 90% sequence identity thereto, optionally wherein the alpha chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, and the beta chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 25, 26 and 27, respectively; or b) the alpha chain and the beta chain each comprise the amino acid sequences of SEQ ID NOs: NO: Amino acid sequence of 6 and 21.

80. A TCR or antigen-binding fragment thereof according to any one of embodiments 74, 76 and 77, wherein: the Cα region comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 29 or an amino acid sequence having at least 90% sequence identity therewith.

81. A TCR or antigen-binding fragment thereof according to any one of embodiments 74, 76 and 77, wherein: the Cα region comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 350 or an amino acid sequence having at least 90% sequence identity therewith.

82. A TCR or antigen-binding fragment thereof according to any one of embodiments 77 to 81, wherein the introduction of one or more cysteine residues comprises replacing a non-cysteine residue with a cysteine residue.

83. The TCR or antigen-binding fragment thereof according to any one of embodiments 81 to 88, wherein the Cα region comprises cysteine at a position corresponding to position 48 as shown in any one of SEQ ID NOs: 333-337 or at a position corresponding to position 49 as shown in SEQ ID NOs: 338 or 341; and/or the Cβ region comprises cysteine at a position corresponding to position 57 as shown in SEQ ID NOs: 339 or 340 or at a position corresponding to position 58 as shown in SEQ ID NOs: 342 or 343.

84. A TCR or antigen-binding fragment thereof according to any one of embodiments 62 to 83, wherein: a) the alpha chain comprises the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and/or the beta chain comprises the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence having at least 90% sequence identity thereto, wherein the alpha chain comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 10, 11 and 12, and the beta chain comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NO: 25, 26 and 27; or b) the alpha chain and the beta chain respectively comprise the amino acid sequences of SEQ ID NO: NO: Amino acid sequence of 5 and 20.

85. The TCR or antigen-binding fragment thereof of any one of embodiments 62 to 83, wherein: a) the alpha chain comprises the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and/or the beta chain comprises the amino acid sequence shown in SEQ ID NO: 369, or an amino acid sequence having at least 90% sequence identity thereto, optionally wherein the alpha chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, and the beta chain comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 25, 26 and 27, respectively; or b) the alpha chain and the beta chain comprise the amino acid sequences of SEQ ID NOs: NO: Amino acid sequence of 5 and 369.

86. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 85, wherein the TCR or antigen-binding fragment thereof is encoded by a codon-optimized nucleotide sequence.

87. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 86, wherein the α chain and/or the β chain further comprises a signal peptide.

88. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 87, which is isolated, purified or recombinant.

89. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 88, which is human.

90. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 89, which is monoclonal.

91. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 90, wherein the TCR or antigen-binding fragment is a single chain.

92. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 91, wherein the TCR or antigen-binding fragment thereof comprises two chains.

93. A TCR or antigen-binding fragment thereof according to any one of embodiments 1 to 92, wherein the antigen specificity is at least partially CD8-independent.

94. A TCR or antigen-binding fragment as in any one of embodiments 7 to 37 and 41 to 61, wherein the MHC molecule is an HLA-A2 molecule.

95. A nucleic acid molecule encoding a TCR or an antigen-binding fragment thereof or an α chain or β chain thereof according to any one of Examples 1 to 94.

96. A nucleic acid molecule as in Example 95, wherein the nucleotide sequence is codon optimized.

97. A nucleic acid molecule as in Example 95 or Example 96, wherein the nucleotide sequence encoding the α chain and the nucleotide sequence encoding the β chain are separated by a peptide sequence that causes ribosome skipping.

98. A nucleic acid molecule as in Example 97, wherein the peptide that causes ribosome skipping is a P2A or T2A peptide and/or comprises the amino acid sequence shown in SEQ ID NO: 32 or 302.

99. A nucleic acid according to any one of embodiments 95 to 98, wherein the nucleic acid is synthetic.

100. A nucleic acid according to any one of embodiments 95 to 99, wherein the nucleic acid is cDNA.

101. A vector comprising a nucleic acid according to any one of embodiments 95 to 100.

102. A carrier according to embodiment 101, wherein the carrier is a performance carrier.

103. A vector as in Example 101 or Example 102, wherein the vector is a viral vector.

104. A vector as in Example 103, wherein the viral vector is a retroviral vector.

105. A vector according to Example 103 or Example 104, wherein the viral vector is a lentiviral vector.

106. A vector as in Example 105, wherein the lentiviral vector is derived from HIV-1.

107. An engineered cell comprising a nucleic acid molecule as described in any one of Examples 95 to 100 or a vector as described in any one of Examples 101 to 106.

108. An engineered cell comprising a TCR or antigen-binding fragment thereof as described in any one of Examples 1 to 94.

109. An engineered cell as in Example 107 or Example 108, wherein the TCR or antigen-binding fragment thereof is heterologous to the cell.

110. An engineered cell according to any one of Examples 107 to 109, wherein the engineered cell is a cell line.

111. An engineered cell according to any one of embodiments 107 to 110, wherein the engineered cell is a primary cell obtained from an individual.

112. The engineered cell of Example 111, wherein the individual is a mammalian individual.

113. The engineered cell of Example 111 or Example 112, wherein the individual is a human.

114. An engineered cell according to any one of Examples 111 to 113, wherein the engineered cell is a T cell.

115. The engineered cell of Example 114, wherein the T cell is CD8+.

116. The engineered cell of Example 114, wherein the T cell is CD4+.

117. An engineered cell according to any one of Examples 107 to 116, wherein: (a) the TCR or antigen-binding fragment thereof comprises a human α homeostatic (Cα) region and a human β homeostatic (Cβ) region and the engineered cell comprises or expresses an endogenous human TCR; (b) the engineered cell comprises or expresses an endogenous human TCR α chain protein and/or an endogenous TCR β chain protein; and/or (c) the engineered cell comprises a nucleotide sequence encoding a fully endogenous human TCR α chain and/or a fully endogenous human TCR β chain.

118. An engineered cell according to any one of embodiments 107 to 117, wherein the Vα region of the TCR or antigen-binding fragment thereof comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 48, 49 and 50, and the Vβ region of the TCR or antigen-binding fragment thereof comprises CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 61, 62 and 63.

119. An engineered cell according to any one of embodiments 107 to 118, wherein the Vα region of the TCR or antigen-binding fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 47 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; and the Vβ region comprises the amino acid sequence shown in SEQ ID NO: 60 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

120. An engineered cell according to any one of Examples 107 to 119, wherein the Vα region and the Vβ region of the TCR or antigen-binding fragment thereof comprise the amino acid sequences of SEQ ID NOs: 47 and 60, respectively.

121. An engineered cell according to any one of embodiments 107 to 120, wherein: the Cα region of the TCR or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region of the TCR or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 350 or an amino acid sequence having at least 90% sequence identity therewith.

122. The engineered cell of any one of embodiments 107 to 121, comprising gene disruption of the T cell receptor alpha homeostatic ( TRAC ) gene and/or the T cell receptor beta homeostatic ( TRBC ) gene.

123. The engineered cell of embodiment 122, wherein the TRBC gene is one or both of T cell receptor beta homeostasis 1 ( TRBC1 ) or T cell receptor beta homeostasis 2 ( TRBC2 ) genes.

124. A method for producing a cell as in any one of Examples 107 to 123, comprising introducing a vector as in any one of Examples 84 to 89 into the cell in vitro or ex vivo.

125. The method of embodiment 124, wherein the vector is a viral vector and the introduction is performed by transduction.

126. The method of embodiment 124 or embodiment 125, further comprising introducing one or more agents into the cell, wherein each of the one or more agents is independently capable of inducing gene disruption of the T cell receptor alpha homeostatic ( TRAC ) gene and/or the T cell receptor beta homeostatic ( TRBC ) gene.

127. A method as in any one of embodiment 126, wherein the one or more agents capable of inducing gene disruption comprise a DNA-binding protein or a DNA-binding nucleic acid that specifically binds or hybridizes to a target site.

128. The method of embodiment 127, wherein the one or more agents capable of inducing gene disruption comprise (a) a fusion protein comprising a DNA targeting protein and a nuclease or (b) an RNA-guided nuclease.

129. The method of embodiment 128, wherein the DNA-targeting protein or RNA-guided nuclease comprises a zinc finger protein (ZFP), a TAL protein, or a clustered regularly interspaced short palindromic nucleic acid (CRISPR)-associated nuclease (Cas) specific for a target site within the TRAC and/or TRBC gene.

130. The method of embodiment 129, wherein the one or more agents comprise a zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR-Cas9 combination that specifically binds to the target site, recognizes the target site, or hybridizes with the target site.

131. The method of embodiment 129 or embodiment 130, wherein each of the one or more agents comprises a guide RNA (gRNA) having a targeting domain that is complementary to the at least one target site.

132. A method as in Example 131, wherein the one or more agents are introduced as a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas9 protein.

133. The method of embodiment 132, wherein the RNP is introduced via electroporation, particle gun, calcium phosphate transfection, cell compression or extrusion.

134. The method of Example 132 or Example 133, wherein the RNP is introduced via electroporation.

135. A method as in Example 131, wherein the one or more agents are introduced as one or more polynucleotides encoding the gRNA and/or Cas9 protein.

136. An engineered cell comprising a recombinant human TCR containing a human α homeostatic (Cα) region and a human β homeostatic (Cβ) region, and the engineered cell comprises or expresses an endogenous human TCR.

137. The engineered cell of Example 136, wherein the recombinant human TCR comprises a Vα region comprising CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 48, 49 and 50; and a Vβ region comprising CDR-1, CDR-2 and CDR-3, which comprise the amino acid sequences of SEQ ID NOs: 61, 62 and 63.

138. An engineered cell as in Example 136 or Example 137, wherein the recombinant human TCR comprises a Vα region comprising the amino acid sequence shown in SEQ ID NO: 47 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith; and a Vβ region comprising the amino acid sequence shown in SEQ ID NO: 60 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity therewith.

139. An engineered cell according to any one of Examples 136 to 138, wherein the recombinant human TCR comprises a Vα region and a Vβ region, which respectively comprise the amino acid sequences of SEQ ID NOs: 47 and 60.

140. An engineered cell according to any one of embodiments 136 to 139, wherein the recombinant human TCR comprises a Cα region comprising the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity therewith; and/or a Cβ region comprising the amino acid sequence shown in SEQ ID NO: 350 or an amino acid sequence having at least 90% sequence identity therewith.

141. A method for producing an engineered cell as in any one of Examples 107 to 123 and 136 to 140, comprising introducing a vector as in any one of Examples 108 to 113 into a cell in vitro or ex vivo.

142. The method of embodiment 141, wherein the vector is a viral vector and the introduction is performed by transduction.

143. A composition comprising an engineered cell according to any one of Examples 107 to 123 and 136 to 140.

144. A composition as in Example 143, wherein the engineered cells comprise CD4+ and/or CD8+ T cells.

145. A composition according to Example 144 or Example 145, wherein the engineered cells comprise CD4+ and CD8+ T cells.

146. A composition comprising the engineered CD8+ cells of Example 115 and the engineered CD4+ cells of Example 116.

147. A composition as described in any one of embodiments 143 to 146, wherein the TCR or antigen-binding fragment thereof binds to or recognizes a peptide antigenic determinant of HPV 16 in the context of an MHC molecule at least partially in a CD8-independent manner.

148. A composition as in any one of embodiments 143 to 147, wherein the CD8+ cells and CD4+ cells are engineered with the same TCR or antigen binding fragment thereof and/or each is engineered with a TCR or antigen binding fragment thereof that binds to or recognizes the same peptide antigenic determinant of HPV 16 in the context of an MHC molecule.

149. A composition according to any one of Examples 143 to 148, further comprising a pharmaceutically acceptable excipient.

150. A method of treatment comprising administering an engineered cell of any one of Examples 107 to 123 and 136 to 140 to an individual suffering from a disease or condition associated with HPV.

151. A method of treatment comprising administering a composition of any one of Examples 143 to 149 to an individual suffering from a disease or condition associated with HPV.

152. The method of embodiment 150 or embodiment 151, wherein the disease or condition is related to HPV16.

153. The method of any one of embodiments 150 to 152, wherein the disease or condition is cancer.

154. A method of treatment comprising administering to an individual suffering from cancer an engineered cell according to any one of embodiments 107 to 123 and 136 to 140.

155. A method of treatment comprising administering a composition of any one of Examples 143 to 149 to a subject suffering from cancer.

156. The method of any one of embodiments 151 to 155, wherein the individual is a human.

157. A composition according to any one of embodiments 143 to 149, which is used to treat a disease or condition associated with HPV in an individual.

158. Use of a composition according to any one of embodiments 143 to 149 for the manufacture of a medicament for treating a disease or condition associated with HPV in an individual.

159. The composition of Example 157 or the use of Example 158, wherein the disease or condition is related to HPV16.

160. A composition or use according to any one of embodiments 157 to 159, wherein the disease or condition is cancer.

161. A composition according to any one of embodiments 143 to 149, which is used to treat cancer in an individual.

162. Use of a composition according to any one of embodiments 143 to 149 for the manufacture of a medicament for treating cancer in an individual.

163. A composition or use according to any one of embodiments 157 to 162, wherein the individual is a human.

IX. Examples

The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1: Screening and selection of HPV-16 E6 and E7 antigen-specific T cell receptors from donors

Screening methods using autologous dendritic cells and T cells generate antigen-specific T cell receptors (TCRs) that specifically bind to human papillomavirus 16 (HPV16) E6 (29-38) or E7 (11-19) peptides presented on MHC-I molecules and survive and/or are enriched over time after multiple rounds of antigen stimulation. Clonal T cell lines are generated and assessed for binding to peptide-MHC tetramers and cytolytic activity. High-throughput paired TCR sequencing is then used to determine the sequences of individual paired TCR alpha and beta chains in the pure T cell lines with observed binding or functional activity on a single cell basis.

A. Generation and selection of human HPV-specific T cells and TCRs

Briefly, peptide-pulsed antigen-presenting cells were generated from peripheral blood mononuclear cells (PBMCs). Specifically, peptide-pulsed HLA:A02:01APCs were generated with either the HPV 16 E6(29-38) peptide (TIHDIILECV; SEQ ID NO: 268) or the E7(11-19) peptide (YMLDLQPET; SEQ ID NO: 271). Autologous CD8+ T cells from normal human donors (or donors with HPV+ head and neck cancer in the case of the identified TCRs designated TCR 56 and TCR 65) were cultured with the peptide-pulsed cells for multiple rounds and selected based on binding to peptide-loaded autologous MHC tetramers. Typically, cells underwent two rounds of stimulation in the presence of peptide-pulsed cells (peptide concentration was maintained at 100 ng/mL during both rounds).

After two rounds of stimulation, cells were sorted by flow cytometry into populations that were positive or negative for binding to peptide-MHC tetramers containing the appropriate tetramers. After repeated stimulation, cell populations that stained positively for peptide-loaded autologous MHC tetramers were identified by flow cytometry.

B. Sequencing

The above tetramer binding-based selected cell populations undergo high-throughput single-cell sequencing for TCR alpha and beta chain pairs. High-throughput single-cell TCR sequencing is performed generally as described in published PCT patent application publications WO2012/048340, WO2012/048341, and WO2016/044227. The sequencing method employs single-cell droplets and samples and molecular barcodes to identify individual pairs of TCR alpha and beta chain sequences at the single cell level for each of a large number (e.g., millions) of single cells present in a single starting composition, and to assess the abundance of each TCR pair in each population assessed. The ability to identify and quantify TCR pairs at the single cell level allows for the assessment of the frequency of each of each TCR pair in each of the individual positive and negative fractions, and for the assessment of the enrichment and persistence of TCRs during multiple rounds of antigen stimulation. TCR pairs identified in this analysis were selected based on their presence in the peptide-bound fraction after rounds 2 and 3, their greater abundance in the positive fraction compared to the negative fraction in each of these rounds, and their enrichment over time after multiple rounds of antigen exposure.

Nucleotide sequences encoding the beta and alpha chains, respectively, of the cloned TCR are separated by a sequence encoding a P2A polypeptide and inserted into a vector, such as a lentiviral vector, for expression of the TCR chain in T cell lines and primary T cells using standard methods. The TCR is cloned to contain mouse constant regions containing added cysteines in the alpha and beta chains to create engineered disulfide bonds. The TCR chain is modified by changing residue 48 in the Cα region from Thr to Cys and residue 57 in the Cβ region from Ser to Cys to promote non-native disulfide bonds (see Kuball et al. (2007) Blood, 109: 2331-2338). The lentiviral construct also contains sequences encoding a truncated receptor as a surrogate marker for transduction and expression, separated from the recombinant TCR encoding sequence by a sequence encoding the T2A ribosomal skipping sequence.

C. Functional assessment of cells transduced with T cell receptors

Primary CD4+ and CD8+ T cells are transduced with lentiviral vector particles encoding the selected TCR. Functional activities of these transduced T cells are assessed, such as the ability to specifically bind antigen and to exhibit lytic activity in response to cells displaying peptide:MHC.

CD8+ and CD4+ T cells were isolated from normal human donors and stimulated with anti-CD3/anti-CD28 reagents and subsequently transduced with lentiviral preparations encoding various TCRs or underwent mock transduction control treatments (cells were treated under the same conditions as used for lentiviral transduction, but without the addition of lentivirus). After transduction, cells were cultured in medium containing human serum and interleukins.

After transduction, cells were assessed by flow cytometry for staining with antibodies recognizing surrogate markers, anti-CD8 antibodies, and HPV 16 E6(29-38)- or HPV16 E7(11-19)-peptide-MHC tetramer complexes. Cells expressing reference TCRs known to bind to exemplary E6(29-38)- or exemplary E7(11-19)-specific TCRs (described in International PCT Publication No. WO 2015/184228) were also assessed in this study. The reference TCRs contained mouse constant regions.

TCR-transduced primary T cells, including a reference control, were also assessed for their ability to lyse target cells in an antigen-specific manner using cancer cell lines expressing the antigen. TCR-transduced T cells were incubated with the squamous cell carcinoma cell line UPCI:SCC152 (ATCC® CRL-3240 ^™ ) at various effector:target (E:T) ratios, including 10:1, 5:1, and 2.5:1. Cultures containing target cells that did not express a TCR with cytolytic activity ("negative") were used as negative controls. T cells expressing TCR49, an E7(11-19)-specific TCR, were also tested. Target SCC152 cells were labeled with NucLight Red (NLR) to allow tracking of the cells by microscopy. Cytolytic activity was assessed by measuring the loss of live target cells over a two-day period over approximately 48 to 72 hours, as determined by red fluorescent signal (using the IncuCyte® Live Cell Analysis System, Essen Bioscience). Cytolytic activity of exemplary TCR-transduced T cells against SCC152 target cells at an E:T ratio of 10:1 is shown in FIG1 . The cytolytic activity of T cells expressing TCR 57 at each of the assessed E:T ratios of 10:1 (high), 5:1 (medium), and 2.5:1 (low) is shown in Figure 2 , compared to a reference TCR known to bind the same peptide epitope. As shown, T cells expressing TCR 57 still exhibited substantial cytolytic activity even at the lowest E:T ratio assessed.

The results of the binding and cytolytic activity studies of exemplary identified TCRs are summarized in Table E1 . Rows 3 and 4 show the percentage of CD8+ cells and CD8- cells (CD4+ cells) that stain positive for the E7 (11-19) specific tetramer in flow cytometry. Rows 5 and 6 show the percentage of CD8+ cells and CD8- cells (CD4+ cells) that stain positive for the E6 (29-38) specific tetramer in flow cytometry. As shown, TCR 57 exhibits CD8-independent binding. Row 7 of the table summarizes the binding activity to the relevant peptides from rows 3 to 6, indicating the ability of the TCRs expressed by primary T cells to specifically bind to the peptides in the context of HLA:A02:01. As shown in row 8, four TCR-expressing T cells (TCR 56, TCR 57, TCR63, and TCR64) and a reference control TCR-expressing T cell were observed to exhibit lytic activity against target cells presented with the relevant HPV16-derived peptides in the context of HLA-A02:01. Interestingly, lytic activity was observed in this analysis for TCR-expressing cells that did not exhibit detectable binding to tetramers as measured by flow cytometry.

D. Exemplary Identified TCRs

The identified TCR was also reformatted in a fully human format containing a human constant domain chain. Similar to the mouse constant domain sequence, the TCR chain was modified by changing residue 48 from Thr to Cys in the Cα region and residue 57 from Ser to Cys in the Cβ region to include mutations that promote the formation of non-native disulfide bonds at the interface between the TCR constant domains to increase the pairing and stability of the TCR (see Kuball et al. (2007) Blood, 109: 2331-2338).

Table E2 lists exemplary E6 (29-38) and E7 (11-19) specific TCRs isolated, evaluated and sequenced using the methods described above. Sequence identifiers (SEQ ID NO:) indicating nucleotide and amino acid sequences of the alpha and beta chains containing human or mouse constant region sequences. The table also lists sequence identifiers (SEQ ID NO:) corresponding to exemplary full-length encoding amino acid sequences containing the beta and alpha chain sequences of each corresponding TCR separated by a sequence encoding a ribosomal skipping P2A sequence (the P2A linker shown in SEQ ID NO: 32, encoded by the nucleotides shown in SEQ ID NO: 31) (designated "β-P2A-α"). In some cases, TCR 57 and/or other TCRs are alternatively formatted with the human beta constant region shown in SEQ ID NO: 350.

In some cases, the nucleotide sequence encoding the TCR generated as described above is modified by codon optimization. The resulting modified nucleotide sequence and the corresponding SEQ ID NO corresponding to the encoded amino acid sequence for the modified version of each TCR are shown in Table E3 . For each TCR modified as described above, constructs containing modified nucleotide sequences encoding the variable regions of the beta and alpha chains of the cloned TCR were generated, separated by sequences encoding the P2A polypeptide, also provided in the table.

Example 2: Evaluation of the Expression and Activity of Exemplary Recombinant T Cell Receptors (TCRs)

A polynucleotide encoding exemplary TCR 57 (formatted with cysteine modifications on the human constant region as described in Example 1D, and in which the sequences encoding the TCR α and TCR β chains are separated by a 2A ribosomal skipping element) was incorporated into an exemplary HIV-1 derived lentiviral vector. Pseudotyped lentiviral vector particles were generated by transiently transfecting HEK-293T cells with the resulting vector, a helper plasmid (containing a gagpol plasmid and a rev plasmid), and a pseudotype plasmid by standard procedures, and used to transduce cells.

Primary human CD4+ and CD8+ T cells were isolated from 5 independent donors by an immunoaffinity-based method. The isolated CD8+ and CD4+ T cells were stimulated with anti-CD3/anti-CD28 reagents, respectively, and then transduced with lentiviral vectors encoding TCRs, respectively. As a control, cells were transduced with a lentiviral vector encoding a reference E7(11-19)-specific TCR (containing a mouse constant region; International PCT Publication No. WO 2015/184228). After transduction, cells were cultured in a medium containing human serum and cytokines. Cells were assessed for cytolytic activity and the ability to induce cytokine production.

A. Reference TCR

For this study, activity was compared to a reference E7(11-19)-specific TCR that binds E7(11-19) and exhibits activity in response thereto and includes a mouse constant region (International PCT Publication No. WO 2015/184228). To assess whether the antigen binding region of the reference TCR would exhibit E7(11-19) antigen-specific activity when included in a TCR containing a human constant region, the α-chain and β-chain of the reference TCR were replaced with fully human α-chain and β-chain constant regions, respectively. The human constant regions used included a mutation from Thr to Cys at residue 48 in the Cα region and a mutation from Ser to Cys at residue 57 in the Cβ region (see Kuball et al. (2007) Blood, 109: 2331-2338), as described above. Primary human CD8+ T cells were transduced with lentiviral vector particles encoding TCRs containing reference TCR variable regions and mouse constant regions or human constant regions, or underwent mock transduction control treatment (cells were treated under the same conditions as those used for lentiviral transduction, but no lentivirus was added).

Primary human T cells transduced with TCRs containing reference TCR variable regions were assessed for their ability to lyse target cells in an antigen-specific manner using cancer cell lines expressing the antigen. TCR-transduced T cells were incubated with the squamous cell carcinoma cell line UPCI:SCC152 (ATCC® CRL-3240 ^™ ) at a 5:1 effector:target (E:T) ratio. Target SCC152 cells were labeled with NucLight Red (NLR) to allow tracking of the cells by microscopy. Cytolytic activity was assessed by measuring the loss of live target cells over a two-day period over a period of approximately 46 hours, as determined by red fluorescent signal (using the IncuCyte® Live Cell Analysis System, Essen Bioscience). As shown in Figure 3 , T cells expressing TCRs including variable regions of reference TCRs exhibited cytolytic activity when such TCRs included mouse constant regions, but not when such TCRs included human constant regions. The results are consistent with the negative effects of endogenous human TCR chains on the expression, activity and/or function of recombinant TCRs including variable regions of reference TCRs when expressed in human T cells, and that such effects can be mitigated by including a combination of mouse constant regions and reference TCR variable regions (International PCT Publication No. WO 2015/184228).

B. Cytolytic activity and interleukin production

Primary human CD8+ T cells and primary human CD4+ T cells were individually engineered to express TCR 57 (containing human constant regions) or reference TCR (containing mouse constant regions) as described in Example 2A above, and their antigen-specific activity was assessed. Cells expressing the corresponding recombinant TCR (effector cells) were cultured with target cells expressing HPV 16 E7 labeled with NucLight Red (NLR). The ability of T cells to antigen-specifically lyse target cells was assessed by measuring the loss of labeled target cells every 2 hours for 48 hours after co-culture. The area under the curve (AUC) of the killing % was calculated. Interleukin production was assessed by measuring interferon-γ (IFNγ) by ELISA in supernatants collected 48 hours after co-culture with target cells as described above. For comparison with T cells expressing a reference TCR, the killing AUC and interleukin production of T cells expressing the reference TCR (CD8+ or CD4+) were normalized to the killing observed with T cells expressing TCR 57 (CD8+ or CD4+), and the fold change was determined.

Results for CD8+ T cells expressing TCRs are shown in FIG4A and results for CD4+ T cells expressing TCRs are shown in FIG4B . As shown, CD8+ T cells expressing TCR 57 and CD4+ T cells expressing TCR 57 exhibit cytolytic activity against target cells and antigen-specific cytokine production. The results are at least comparable to the activity observed for T cells expressing a reference TCR (containing a mouse constant region). The activity observed in CD4+ T cells is consistent with the ability of cells expressing TCR 57 or a TCR with its antigen binding region to exhibit antigen-specific activity in a CD8-independent manner. Furthermore, the results demonstrate that human T cells expressing recombinant TCRs having an antigen binding region of TCR 57 and including a human constant region, unlike the variable region of the reference TCR, are able to exhibit such activity in human T cells. These results are consistent with the finding that the capacity of recombinant TCRs containing the variable region of TCR 57 is not negatively affected by the presence of endogenous human TCR chains to the extent observed for TCRs containing other variable regions, such as the variable region of the reference TCR. In some embodiments, the ability of the TCRs provided herein to be less affected by endogenous human TCR chains when expressed in human cells or the ability of such cells to maintain activity in the presence of endogenous TCR chains can be attributed to the ability of the provided TCRs to better compete with endogenous TCRs for signaling components, such as CD3 signaling components. In some embodiments, such abilities can be conferred by one or more properties of the TCR, such as sequence-specific properties, for example, sequence-specific properties of the variable region and/or antigen binding region of the TCR.

Example 3: Evaluation of the anti-tumor effects of T cells engineered to express recombinant T cell receptors (TCRs) in vivo in mice

The anti-tumor activity of primary human CD4+ and primary human CD8+ T cells expressing recombinant TCRs containing the variable region of TCR 57 (and containing a human constant region with cysteine modifications) was assessed in vivo by administering the engineered cells in a mouse tumor model. The anti-tumor activity of these cells was compared to the activity of human T cells engineered to express the E7 (11-19) reference TCR (containing a mouse constant region, described in Example 2A above; International PCT Publication No. WO 2015/184228). The mouse tumor model was generated by subcutaneous injection of squamous cell carcinoma cell line UPCI: SCC152 (ATCC® CRL-3240 ^™ ) cells in female NOD/SCID/IL-2Rγ ^null (NSG) mice. Approximately 3 weeks after injection, when tumor-bearing mice exhibited an average tumor volume of approximately 150 mm ³ , mice were divided into groups (n=7 mice/group). 3×10 ⁶ or 1.5×10 ⁶ TCR-expressing cells were administered intravenously to each group of mice. In this study, average tumor volume was assessed twice a week for up to 80 days after administration of the engineered cells.

The results are shown in Figure 5A (mean tumor volume) and Figure 5B (tumor volume of individual mice in each group). Primary human T cells expressing recombinant TCR 57 exhibited anti-tumor activity in this model at least comparable to cells expressing a recombinant reference TCR containing a mouse constant region, as demonstrated by essentially complete reduction of tumor volume until at least day 80, the last time point assessed at both cell doses tested in this study. The results are consistent with the observation that T cells engineered to express a TCR containing a human constant region and having an antigen binding region of TCR 57 are able to exhibit substantial anti-tumor activity in the presence of endogenous human TCRs.

Example 4: Evaluation of the performance of exemplary recombinant T cell receptors (TCRs) compared to reference TCRs

The cell surface expression of exemplary anti-human papillomavirus 16 (HPV16) E7 (11-19) T cell receptors (TCRs) was assessed and compared to the cell surface expression of reference anti-HPV16 TCRs.

Primary human CD4+ and CD8+ T cells were isolated from independent donors by an immunoaffinity-based approach, stimulated with anti-CD3/anti-CD28 reagents, and subsequently transduced with lentiviral vectors encoding exemplary TCRs (containing variable regions from TCR 57 and human constant regions) as generally described above in Example 2. As a control, cells were transduced with a lentiviral vector encoding a reference E7(11-19)-specific TCR (containing the mouse constant regions described above in Example 2A). After transduction and culture, cells were assessed by flow cytometry for cell surface expression of recombinant TCRs and CD8 dependence by staining with anti-Vβ2 antibody (for TCR 57) or anti-Vβ22 antibody (for reference TCR), HPV16 E7(11-19)-peptide-MHC tetramer complexes, and anti-CD8 antibody.

As shown in Figures 6A-6B , the exemplary TCR (containing the variable region from TCR 57 and the human constant region) was expressed on the cell surface and bound to the E7 (11-19) peptide-MHC tetramer complex to an extent comparable to that of a reference TCR containing a mouse constant region. Using cells from 3 or more independent donors, an average of approximately 50-60% of the transduced cells exhibited cell surface expression of the TCR. The results show that even in the presence of endogenous human TCR chains, there is high expression and specific tetramer binding by CD4+ and CD8+ cells expressing the exemplary TCR.

Example 5: Assessing the Activity of an Exemplary Recombinant T Cell Receptor (TCR) Compared to a Reference TCR

The cytolytic activity and interleukin secretion of exemplary anti-human papillomavirus 16 (HPV16) E7 (11-19) T cell receptors (TCRs) were assessed and compared to those of reference anti-HPV16 TCRs.

Primary human CD4+ and CD8+ T cells from three independent donors engineered to express TCR 57 or a reference TCR as a control (described in Examples 3 and 4 above) were assessed for antigen-specific cytolytic activity and interleukin production. Cells expressing exemplary TCRs (effector cells) were cultured with various NucLight Red (NLR)-labeled target cells at effector:target (E:T ⁾ ratios of 1:25:1, 2.5:1, ⁵ : ^{1 ,} or 10:1 ... The ability of T cells to antigen-specifically lyse target cells was assessed by measuring the loss of labeled target cells as determined by red fluorescent signal (using the IncuCyte® Live Cell Analysis System, Essen Bioscience). The area under the curve (AUC) of % kill was calculated, and the results were normalized to the AUC of the mock control. Interleukin production was assessed by measuring interferon-γ (IFNγ), tumor necrosis factor α (TNFα), and interleukin-2 (IL-2), generally as described above in Example 2B. As controls, cytolytic activity and cytokine production were also assessed after incubation with K562 cells expressing HPV16 E6 (non-specific antigen) or SiHa squamous cell carcinoma cells (ATCC® HTB-35 ^™ ) expressing HPV16 E7 but not HLA-A02:01.

As shown in Figures 7A to 7D , CD8+ ( Figures 7A to 7C ) and CD4+ ( Figures 7B and 7D ) T cells expressing TCR 57 (an exemplary anti-HPV16 E7 TCR) exhibited antigen-specific cytotoxicity against various target cells at various E:T ratios tested, to a similar extent as cells expressing a reference TCR containing a mouse constant region. As shown in Figure 7E , after co-culture with various types of target cells expressing HPV16 E7, both CD8+ and CD4+ T cells produced IFNγ, generally to a similar extent as cells expressing a reference TCR containing a mouse constant region. Similar results were observed for TNFα and IL-2 production.

Results were consistent with antigen- and HLA subtype-specific cytolytic activity and interleukin production of T cells engineered to express TCR 57 (containing a human constant region), including strong activity of the exemplary TCR at low E:T ratios. Results also showed that engineered CD4+ cells themselves could promote cytolytic activity in addition to interleukin production to aid CD8+ T cells.

Example 6: Evaluation of the anti-tumor effects of CD4+ or CD8+ T cells engineered to express exemplary recombinant T cell receptors (TCRs) in mice in vivo

The anti-tumor activity of CD4+ and CD8+ primary T cells from human donors engineered to express an exemplary TCR with cysteine modifications (containing the variable region of TCR 57 and a human constant region) was assessed in vivo by administering these engineered cells in an SCC152 tumor xenograft mouse model, generally as described in Example 3 above, with the following exceptions: only 3×10 ⁶ engineered CD8+ cells or only 3×10 ⁶ engineered CD4+ cells were administered per mouse. Mean tumor volume was assessed up to 80 days after administration of the engineered cells and compared to that in mice receiving either tumor xenografts alone (Tumor Alone) or mock-transduced T cells (Mock).

As shown in Figure 8 , mice administered primary human CD4+ cells alone or engineered primary human CD8+ cells engineered to express an exemplary TCR (containing the variable region from TCR 57 and the human constant region) alone exhibited essentially complete reduction of tumor size. The results are consistent with the observation that both CD4+ cells and CD8+ cells promote anti-tumor activity in mice.

Example 7: Evaluation of target specificity of exemplary recombinant T cell receptors (TCRs)

Evaluation of target binding specificity and human leukocyte antigen (HLA) subtype specificity of exemplary anti-HPV16 E7 (11-19) TCRs.

A. Peptide scanning

A synthetic peptide scanning library was generated to assess the binding motifs of the epitopes bound by the exemplary anti-HPV16 E7 (11-19) TCR (containing the variable region from TCR 57). A 180-member peptide library was synthesized in which all 20 amino acids were present in all 9 peptide positions of HPV16 E7 (11-19) (YMLDLQPET; SEQ ID NO: 271). For antigen presentation, T2 (174 x CEM.T2) lymphoblastoid cells (ATCC® CRL-1992 ^™ ) expressing the major histocompatibility complex (MHC) type HLA-A02:01 and lacking endogenous peptides were pulsed with synthetic peptides from the library. Peptide-pulsed T2 cells were co-cultured with a Jurkat T reporter cell line expressing TCR 57 and containing a luminescent reporter induced by activation of signals from the T cell receptor, and luminescence was assessed.

The results of the reporter analysis potentially identified the permissible residues at each position of the E7(11-19) peptide, which are shown in Table E4 below.

The human genome was searched for permissive motifs that could be present in endogenous human proteins. 22 candidate peptides were identified and pulsed onto T2 cells and assessed using the Jurkat reporter described above. Two peptides (peptide A and peptide B) exhibited luminescent signals using this assay. Binding of both peptides to TCR 57 was assessed by peptide titration analysis and in silico analysis of affinity to HLA-A02:01 using the Immune Epitope Determinant Database (IEDB).

As shown in Figure 9 , TCR 57 exhibited greater sensitivity to the HPV16 E7 (11-19) peptide than to peptide A and peptide B. Peptide A, but not peptide B, exhibited a relatively higher affinity for HLA-A02:01 as determined by computer simulation analysis.

Engineered reporter T cells expressing TCR 57 were also evaluated for activation signals (via luminescent reporters) and cytolytic activity against target cells (B cells and T cells) known to endogenously express proteins containing peptide A or peptide B. The results showed that TCR 57 did not exhibit activation or cytolytic activity against cells (B cells and T cells) endogenously expressing peptide A or peptide B.

B. HLA subtype specificity

The specificity of the human leukocyte antigen (HLA) subtype recognized by the exemplary TCR (containing the variable region from TCR 57) was assessed using a panel of B-lymphoblastoid cell lines (BLCLs) expressing various common HLA alleles. A panel of over 70 BLCLs expressing over 95 different HLA-A, B, or C alleles, covering 95% of the most common HLA alleles in North American populations, displayed endogenous peptides or were pulsed with the E7(11-19) peptide and co-cultured with the Jurkat T reporter cell line expressing TCR57 described above.

The results showed that TCR 57 showed activity only against cells pulsed with the E7(11-19) peptide expressing HLA-A02:01. No alloreactivity was observed for all other HLA subtypes tested.

The results are consistent with the observation that the exemplary TCR (containing the variable region from TCR 57) does not exhibit substantial off-target activity and does not exhibit reactivity to different HLA subtypes. The results show that the exemplary TCR exhibits specific and sensitive activity against the HPV 16 E7 (11-19) peptide and exhibits reduced or low risk of off-target activity.

The present invention is not intended to be limited in scope to the specifically disclosed embodiments, which are provided, for example, to illustrate various aspects of the present invention. Various modifications to the compositions and methods will become apparent from the description and teachings herein. Such variations may be implemented without departing from the true scope and spirit of the present invention and are intended to be within the scope of the present invention.

<110> Juno Therapeutics, Inc.

<120> HPV-specific binding molecules

<130> 735042015142

<140> TW 108112029

<141> 2019-04-03

<150> US 62/653,516

<151> 2018-04-05

<150> US 62/739,145

<151> 2018-09-28

<160> 424

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 870

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 56 alpha natural with codon optimization

<400> 1

<210> 2

<211> 858

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 56 α natural with codon optimization

<400> 2

<210> 3

<211> 907

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized TCR 56 α using codon-optimized human constants

<400> 3

<210> 4

<211> 895

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized TCR 56 α using codon-optimized mouse constants

<400> 4

<210> 5

<211> 255

<212> PRT

<213> Artificial sequence

<220>

<223> Human invariant TCR 56 α with codon optimization

<400> 5

<210> 6

<211> 251

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 56 α with codon optimization

<400> 6

<210> 7

<211> 289

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 56 α with codon-optimized human constant and signal peptides

<400> 7

<210> 8

<211> 285

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 56 α with codon-optimized mouse constant and signal peptide

<400> 8

<210> 9

<211> 114

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 56 α variable region

<400> 9

<210> 10

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 56 α CDR1

<400> 10

<210> 11

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 56 α CDR2

<400> 11

<210> 12

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 56 α CDR3

<400> 12

<210> 13

<211> 34

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 56 α signal peptide

<400> 13

<210> 14

<211> 141

<212> PRT

<213> Homo sapiens

<220>

<223> α Human Constant Zone

<400> 14

<210> 15

<211> 137

<212> PRT

<213> House mouse

<220>

<223> α mouse homeostasis region

<400> 15

<210> 16

<211> 957

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 56 β natural with codon optimization

<400> 16

<210> 17

<211> 945

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 56 β native with codon optimization

<400> 17

<210> 18

<211> 966

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized TCR 56 β using codon-optimized human constant

<400> 18

<210> 19

<211> 954

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized TCR 56 β using codon-optimized mouse constants

<400> 19

<210> 20

<211> 294

<212> PRT

<213> Artificial sequence

<220>

<223> Human invariant TCR 56 β with codon optimization

<400> 20

<210> 21

<211> 290

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 56 β with codon optimization

<400> 21

<210> 22

<211> 318

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 56 β with codon-optimized human homeostatic and signal peptides

<400> 22

<210> 23

<211> 314

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 56 β with codon-optimized mouse homeostasis and signal peptide

<400> 23

<210> 24

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 56 β variable region

<400> 24

<210> 25

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 56 β CDR1

<400> 25

<210> 26

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 56 β CDR2

<400> 26

<210> 27

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 56 β CDR3

<400> 27

<210> 28

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 56 β signal peptide

<400> 28

<210> 29

<211> 177

<212> PRT

<213> Homo sapiens

<220>

<223> β human homeostasis zone

<400> 29

<210> 30

<211> 173

<212> PRT

<213> House mouse

<220>

<223> β mouse homeostasis region

<400> 30

<210> 31

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> P2A

<400> 31

<210> 32

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> P2A

<400> 32

<210> 33

<211> 1026

<212> DNA

<213> Artificial sequence

<220>

<223> Has the complete natural sequence of human constant TCR 56

<400> 33

<210> 34

<211> 1869

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of mouse constant TCR 56

<400> 34

<210> 35

<211> 1939

<212> DNA

<213> Artificial sequence

<220>

<223> The optimized complete sequence of TCR 56 codons with human constants

<400> 35

<210> 36

<211> 1915

<212> DNA

<213> Artificial sequence

<220>

<223> The optimized complete sequence of mouse constant TCR 56 codons

<400> 36

<210> 37

<211> 629

<212> PRT

<213> Artificial sequence

<223> Has the complete sequence of human-specific TCR 56

<400> 37

<210> 38

<211> 621

<212> PRT

<213> Artificial sequence

<220>

<223> Full sequence of mouse constant TCR 56

<400> 38

<210> 39

<211> 810

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 57 alpha natural with codon optimization

<400> 39

<210> 40

<211> 798

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 57 alpha natural with codon optimization

<400> 40

<210> 41

<211> 810

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized TCR 57 α using codon-optimized human constants

<400> 41

<210> 42

<211> 798

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized TCR 57 α using codon-optimized mouse constants

<400> 42

<210> 43

<211> 251

<212> PRT

<213> Artificial sequence

<220>

<223> Human invariant TCR 57 α with codon optimization

<400> 43

<210> 44

<211> 247

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 57 α with codon optimization

<400> 44

<210> 45

<211> 269

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 57 α with codon-optimized human constant and signal peptides

<400> 45

<210> 46

<211> 265

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 57 α with codon-optimized mouse constant and signal peptide

<400> 46

<210> 47

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 57 α variable region

<400> 47

<210> 48

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 57 α CDR1

<400> 48

<210> 49

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 57 α CDR2

<400> 49

<210> 50

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 57 α CDR3

<400> 50

<210> 51

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 57 α signaling peptide

<400> 51

<210> 52

<211> 924

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 57 β natural with codon optimization

<400> 52

<210> 53

<211> 912

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 57 β native with codon optimization

<400> 53

<210> 54

<211> 924

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized TCR 57 β using codon-optimized human constant

<400> 54

<210> 55

<211> 912

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized TCR 57 β using codon-optimized mouse constants

<400> 55

<210> 56

<211> 293

<212> PRT

<213> Artificial sequence

<220>

<223> Human invariant TCR 57 β with codon optimization

<400> 56

<210> 57

<211> 289

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 57 β with codon optimization

<400> 57

<210> 58

<211> 307

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 57 β with codon-optimized human homeostatic and signal peptides

<400> 58

<210> 59

<211> 303

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 57 β with codon-optimized mouse homeostasis and signal peptide

<400> 59

<210> 60

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 57 β variable region

<400> 60

<210> 61

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 57 β CDR1

<400> 61

<210> 62

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 57 β CDR2

<400> 62

<210> 63

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 57 β CDR3

<400> 63

<210> 64

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 57 β signaling peptide

<400> 64

<210> 65

<211> 1800

<212> DNA

<213> Artificial sequence

<220>

<223> Has the complete natural sequence of human constant TCR 57

<400> 65

<210> 66

<211> 1776

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of mouse constant TCR 57

<400> 66

<210> 67

<211> 1800

<212> DNA

<213> Artificial sequence

<220>

<223> The optimized complete sequence of TCR 57 codons with human constants

<400> 67

<210> 68

<211> 1776

<212> DNA

<213> Artificial sequence

<220>

<223> The complete sequence of TCR 57 codon optimization with mouse constitutiveness

<400> 68

<210> 69

<211> 598

<212> PRT

<213> Artificial sequence

<220>

<223> Has the complete sequence of human-specific TCR 57

<400> 69

<210> 70

<211> 590

<212> PRT

<213> Artificial sequence

<220>

<223> Full sequence of TCR 57 with mouse constitutive identity

<400> 70

<210> 71

<211> 825

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 58 alpha natural with codon optimization

<400> 71

<210> 72

<211> 812

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 58 alpha natural with codon optimization

<400> 72

<210> 73

<211> 252

<212> PRT

<213> Artificial sequence

<220>

<223> Human invariant TCR 58 α with codon optimization

<400> 73

<210> 74

<211> 248

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 58 α with codon optimization

<400> 74

<210> 75

<211> 274

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 58 α with codon-optimized human constant and signal peptides

<400> 75

<210> 76

<211> 270

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 58 α with codon-optimized mouse constant and signal peptide

<400> 76

<210> 77

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 58 α variable region

<400> 77

<210> 78

<211> 6

<212> PRT

<213> Artificial sequence

<223> TCR 58 α CDR1

<400> 78

<210> 79

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 58 α CDR2

<400> 79

<210> 80

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 58 α CDR3

<400> 80

<210> 81

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 58 α signal peptide

<400> 81

<210> 82

<211> 930

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 58 β natural with codon optimization

<400> 82

<210> 83

<211> 918

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 58 β native with codon optimization

<400> 83

<210> 84

<211> 290

<212> PRT

<213> Artificial sequence

<220>

<223> Human invariant TCR 58 β with codon optimization

<400> 84

<210> 85

<211> 286

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 58 β with codon optimization

<400> 85

<210> 86

<211> 309

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 58 β with codon-optimized human homeostatic and signal peptides

<400> 86

<210> 87

<211> 305

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 58 β with codon-optimized mouse homeostasis and signal peptide

<400> 87

<210> 88

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 58 β variable region

<400> 88

<210> 89

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 58, 59, 60, 61 β CDR1

<400> 89

<210> 90

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 58, 59, 60, 61 β CDR2

<400> 90

<210> 91

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 58 β CDR3

<400> 91

<210> 92

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 58, 59, 60, 61 β signal peptide

<400> 92

<210> 93

<211> 1821

<212> DNA

<213> Artificial sequence

<220>

<223> Has the full natural sequence of human constant TCR 58

<400> 93

<210> 94

<211> 1797

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of mouse constant TCR 58

<400> 94

<210> 95

<211> 605

<212> PRT

<213> Artificial sequence

<220>

<223> Has the complete sequence of human-specific TCR 58

<400> 95

<210> 96

<211> 597

<212> PRT

<213> Artificial sequence

<220>

<223> Full sequence of TCR 58 with mouse constitutive identity

<400> 96

<210> 97

<211> 831

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 59 alpha natural with codon optimization

<400> 97

<210> 98

<211> 819

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 59 α natural with codon optimization

<400> 98

<210> 99

<211> 252

<212> PRT

<213> Artificial sequence

<220>

<223> Human invariant TCR 59 α with codon optimization

<400> 99

<210> 100

<211> 248

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 59 α with codon optimization

<400> 100

<210> 101

<211> 276

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 59 α with codon-optimized human constant and signal peptides

<400> 101

<210> 102

<211> 272

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 59 α with codon-optimized mouse constant and signal peptide

<400> 102

<210> 103

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 59 α variable region

<400> 103

<210> 104

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 59 α CDR1

<400> 104

<210> 105

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 59 α CDR2

<400> 105

<210> 106

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 59 α CDR3

<400> 106

<210> 107

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 59 α signal peptide

<400> 107

<210> 108

<211> 930

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 59 β natural with codon optimization

<400> 108

<210> 109

<211> 918

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 59 β native with codon optimization

<400> 109

<210> 110

<211> 290

<212> PRT

<213> Artificial sequence

<220>

<223> Human invariant TCR 59 β with codon optimization

<400> 110

<210> 111

<211> 286

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 59 β with codon optimization

<400> 111

<210> 112

<211> 309

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 59 β with codon-optimized human homeostatic and signal peptides

<400> 112

<210> 113

<211> 305

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 59 β with codon-optimized mouse homeostasis and signal peptide

<400> 113

<210> 114

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 59 β variable region

<400> 114

<210> 115

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 59 β CDR3

<400> 115

<210> 116

<211> 1827

<212> DNA

<213> Artificial sequence

<220>

<223> Has the complete natural sequence of human constant TCR 59

<400> 116

<210> 117

<211> 1803

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of mouse constant TCR 59

<400> 117

<210> 118

<211> 607

<212> PRT

<213> Artificial sequence

<220>

<223> Has the complete sequence of human-specific TCR 59

<400> 118

<210> 119

<211> 599

<212> PRT

<213> Artificial sequence

<220>

<223> Full sequence of TCR 59 with mouse constitutive identity

<400> 119

<210> 120

<211> 837

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 60 alpha natural with codon optimization

<400> 120

<210> 121

<211> 825

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 60 α natural with codon optimization

<400> 121

<210> 122

<211> 256

<212> PRT

<213> Artificial sequence

<220>

<223> Human constant TCR 60 α with codon optimization

<400> 122

<210> 123

<211> 252

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 60 α with codon optimization

<400> 123

<210> 124

<211> 278

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 60 α with codon-optimized human constant and signal peptides

<400> 124

<210> 125

<211> 274

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 60 α with codon-optimized mouse homeostasis and signal peptide

<400> 125

<210> 126

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 60 α variable region

<400> 126

<210> 127

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 60, 61 α CDR1

<400> 127

<210> 128

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 60, 61 α CDR2

<400> 128

<210> 129

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 60 α CDR3

<400> 129

<210> 130

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 60, 61 α signal peptide

<400> 130

<210> 131

<211> 930

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 60 β natural with codon optimization

<400> 131

<210> 132

<211> 918

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 60 β native with codon optimization

<400> 132

<210> 133

<211> 290

<212> PRT

<213> Artificial sequence

<220>

<223> Human constant TCR 60 β with codon optimization

<400> 133

<210> 134

<211> 286

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 60 β with codon optimization

<400> 134

<210> 135

<211> 309

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 60 β with codon-optimized human homeostatic and signal peptides

<400> 135

<210> 136

<211> 305

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 60 β with codon-optimized mouse homeostasis and signal peptide

<400> 136

<210> 137

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 60 β variable region

<400> 137

<210> 138

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 60 β CDR3

<400> 138

<210> 139

<211> 1833

<212> DNA

<213> Artificial sequence

<220>

<223> Has the full natural sequence of human constant TCR 60

<400> 139

<210> 140

<211> 1809

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of mouse constant TCR 60

<400> 140

<210> 141

<211> 609

<212> PRT

<213> Artificial sequence

<220>

<223> Has the full sequence of human-specific TCR 60

<400> 141

<210> 142

<211> 601

<212> PRT

<213> Artificial sequence

<220>

<223> Full sequence of TCR 60 with mouse constants

<400> 142

<210> 143

<211> 828

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 61 alpha natural with codon optimization

<400> 143

<210> 144

<211> 816

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 61 α natural with codon optimization

<400> 144

<210> 145

<211> 253

<212> PRT

<213> Artificial sequence

<220>

<223> Human invariant TCR 61 α with codon optimization

<400> 145

<210> 146

<211> 249

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 61 α with codon optimization

<400> 146

<210> 147

<211> 275

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 61 α with codon-optimized human constant and signal peptides

<400> 147

<210> 148

<211> 271

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 61 α with codon-optimized mouse constant and signal peptide

<400> 148

<210> 149

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 61 α variable region

<400> 149

<210> 150

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 61 α CDR3

<400> 150

<210> 151

<211> 930

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 61 β natural with codon optimization

<400> 151

<210> 152

<211> 918

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 61 β native with codon optimization

<400> 152

<210> 153

<211> 290

<212> PRT

<213> Artificial sequence

<220>

<223> Human constant TCR 61 β with codon optimization

<400> 153

<210> 154

<211> 286

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 61 β with codon optimization

<400> 154

<210> 155

<211> 309

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 61 β with codon-optimized human homeostatic and signal peptides

<400> 155

<210> 156

<211> 305

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 61 β with codon-optimized mouse homeostasis and signal peptide

<400> 156

<210> 157

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 61 β variable region

<400> 157

<210> 158

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 61 β CDR3

<400> 158

<210> 159

<211> 1824

<212> DNA

<213> Artificial sequence

<220>

<223> Has the full natural sequence of human constant TCR 61

<400> 159

<210> 160

<211> 1800

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of mouse constant TCR 61

<400> 160

<210> 161

<211> 606

<212> PRT

<213> Artificial sequence

<220>

<223> Has the complete sequence of human-specific TCR 61

<400> 161

<210> 162

<211> 598

<212> PRT

<213> Artificial sequence

<220>

<223> Full sequence of mouse constant TCR 61

<400> 162

<210> 163

<211> 819

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 62 α natural with codon optimization

<400> 163

<210> 164

<211> 807

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 62 α natural with codon optimization

<400> 164

<210> 165

<211> 252

<212> PRT

<213> Artificial sequence

<220>

<223> Human constant TCR 62 α with codon optimization

<400> 165

<210> 166

<211> 248

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 62 α with codon optimization

<400> 166

<210> 167

<211> 272

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 62 α with codon-optimized human constant and signal peptides

<400> 167

<210> 168

<211> 268

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 62 α with codon-optimized mouse constant and signal peptide

<400> 168

<210> 169

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 62 α variable region

<400> 169

<210> 170

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 62 α CDR1

<400> 170

<210> 171

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 62 α CDR2

<400> 171

<210> 172

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 62 α CDR3

<400> 172

<210> 173

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 62 α signal peptide

<400> 173

<210> 174

<211> 942

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 62 β natural with codon optimization

<400> 174

<210> 175

<211> 930

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 62 β native with codon optimization

<400> 175

<210> 176

<211> 294

<212> PRT

<213> Artificial sequence

<220>

<223> Human constant TCR 62 β with codon optimization

<400> 176

<210> 177

<211> 290

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 62 β with codon optimization

<400> 177

<210> 178

<211> 313

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 62 β with codon-optimized human homeostatic and signal peptides

<400> 178

<210> 179

<211> 309

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 62 β with codon-optimized mouse homeostasis and signal peptide

<400> 179

<210> 180

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 62 β variable region

<400> 180

<210> 181

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 62 β CDR1

<400> 181

<210> 182

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 62 β CDR2

<400> 182

<210> 183

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 62 β CDR3

<400> 183

<210> 184

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 62 β signal peptide

<400> 184

<210> 185

<211> 1827

<212> DNA

<213> Artificial sequence

<220>

<223> Has the full natural sequence of human constant TCR 62

<400> 185

<210> 186

<211> 1803

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of mouse constant TCR 62

<400> 186

<210> 187

<211> 607

<212> PRT

<213> Artificial sequence

<220>

<223> Has the full sequence of human-specific TCR 62

<400> 187

<210> 188

<211> 599

<212> PRT

<213> Artificial sequence

<220>

<223> Full sequence of TCR 62 with mouse constitutive properties

<400> 188

<210> 189

<211> 816

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 63 alpha natural with codon optimization

<400> 189

<210> 190

<211> 804

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 63 α natural with codon optimization

<400> 190

<210> 191

<211> 251

<212> PRT

<213> Artificial sequence

<220>

<223> Human invariant TCR 63 α with codon optimization

<400> 191

<210> 192

<211> 247

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 63 α with codon optimization

<400> 192

<210> 193

<211> 271

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 63 α with codon-optimized human constant and signal peptides

<400> 193

<210> 194

<211> 267

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 63 α with codon-optimized mouse constant and signal peptide

<400> 194

<210> 195

<211> 110

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 63 α variable region

<400> 195

<210> 196

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 63 α CDR1

<400> 196

<210> 197

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 63 α CDR2

<400> 197

<210> 198

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 63 α CDR3

<400> 198

<210> 199

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 63 α signal peptide

<400> 199

<210> 200

<211> 939

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 63 β natural with codon optimization

<400> 200

<210> 201

<211> 927

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 63 β native with codon optimization

<400> 201

<210> 202

<211> 293

<212> PRT

<213> Artificial sequence

<220>

<223> Human constant TCR 63 β with codon optimization

<400> 202

<210> 203

<211> 289

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 63 β with codon optimization

<400> 203

<210> 204

<211> 312

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 63 β with codon-optimized human constant and signal peptides

<400> 204

<210> 205

<211> 308

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 63 β with codon-optimized mouse homeostasis and signal peptide

<400> 205

<210> 206

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 63 β variable region

<400> 206

<210> 207

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 63 β CDR1

<400> 207

<210> 208

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 63 β CDR2

<400> 208

<210> 209

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 63 β CDR3

<400> 209

<210> 210

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 63 β signal peptide

<400> 210

<210> 211

<211> 1821

<212> DNA

<213> Artificial sequence

<220>

<223> Has the full natural sequence of human constant TCR 63

<400> 211

<210> 212

<211> 1797

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of mouse constant TCR 63

<400> 212

<210> 213

<211> 605

<212> PRT

<213> Artificial sequence

<220>

<223> Has the complete sequence of human-specific TCR 63

<400> 213

<210> 214

<211> 597

<212> PRT

<213> Artificial sequence

<220>

<223> Full sequence of mouse constant TCR 63

<400> 214

<210> 215

<211> 840

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 64 alpha natural with codon optimization

<400> 215

<210> 216

<211> 828

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 64 α natural with codon optimization

<400> 216

<210> 217

<211> 257

<212> PRT

<213> Artificial sequence

<220>

<223> Human invariant TCR 64 α with codon optimization

<400> 217

<210> 218

<211> 253

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 64 α with codon optimization

<400> 218

<210> 219

<211> 279

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 64 α with codon-optimized human constant and signal peptides

<400> 219

<210> 220

<211> 275

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 64 α with codon-optimized mouse constant and signal peptide

<400> 220

<210> 221

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 64 α variable region

<400> 221

<210> 222

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 64 α CDR1

<400> 222

<210> 223

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 64 α CDR2

<400> 223

<210> 224

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 64 α CDR3

<400> 224

<210> 225

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 64 α signal peptide

<400> 225

<210> 226

<211> 933

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 64 β natural with codon optimization

<400> 226

<210> 227

<211> 921

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 64 β native with codon optimization

<400> 227

<210> 228

<211> 291

<212> PRT

<213> Artificial sequence

<220>

<223> Human invariant TCR 64 β with codon optimization

<400> 228

<210> 229

<211> 287

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 64 β with codon optimization

<400> 229

<210> 230

<211> 310

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 64 β with codon-optimized human homeostatic and signal peptides

<400> 230

<210> 231

<211> 306

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 64 β with codon-optimized mouse homeostasis and signal peptide

<400> 231

<210> 232

<211> 114

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 64 β variable region

<400> 232

<210> 233

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 64 β CDR1

<400> 233

<210> 234

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 64 β CDR2

<400> 234

<210> 235

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 64 β CDR3

<400> 235

<210> 236

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 64 β signal peptide

<400> 236

<210> 237

<211> 1839

<212> DNA

<213> Artificial sequence

<220>

<223> Has the full natural sequence of human constant TCR 64

<400> 237

<210> 238

<211> 1815

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of mouse constant TCR 64

<400> 238

<210> 239

<211> 611

<212> PRT

<213> Artificial sequence

<220>

<223> Full sequence of TCR 64 with human constants

<400> 239

<210> 240

<211> 603

<212> PRT

<213> Artificial sequence

<220>

<223> Full sequence of mouse constant TCR 64

<400> 240

<210> 241

<211> 828

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 65 alpha natural with codon optimization

<400> 241

<210> 242

<211> 816

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 65 alpha natural with codon optimization

<400> 242

<210> 243

<211> 254

<212> PRT

<213> Artificial sequence

<220>

<223> Human invariant TCR 65 α with codon optimization

<400> 243

<210> 244

<211> 250

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 65 α with codon optimization

<400> 244

<210> 245

<211> 275

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 65 α with codon-optimized human constant and signal peptides

<400> 245

<210> 246

<211> 271

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 65 α with codon-optimized mouse constant and signal peptides

<400> 246

<210> 247

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 65 α variable region

<400> 247

<210> 248

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 65 α CDR1

<400> 248

<210> 249

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 65 α CDR2

<400> 249

<210> 250

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 65 α CDR3

<400> 250

<210> 251

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 65 α signal peptide

<400> 251

<210> 252

<211> 936

<212> DNA

<213> Artificial sequence

<220>

<223> Human constant TCR 65 β natural with codon optimization

<400> 252

<210> 253

<211> 924

<212> DNA

<213> Artificial sequence

<220>

<223> Mouse constant TCR 65 β native with codon optimization

<400> 253

<210> 254

<211> 292

<212> PRT

<213> Artificial sequence

<220>

<223> Human constant TCR 65 β with codon optimization

<400> 254

<210> 255

<211> 288

<212> PRT

<213> Artificial sequence

<220>

<223> Mouse constant TCR 65 β with codon optimization

<400> 255

<210> 256

<211> 311

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 65 β with codon-optimized human homeostatic and signal peptides

<400> 256

<210> 257

<211> 307

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 65 β with codon-optimized mouse homeostasis and signal peptide

<400> 257

<210> 258

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 65 β variable region

<400> 258

<210> 259

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 65 β CDR1

<400> 259

<210> 260

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 65 β CDR2

<400> 260

<210> 261

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 65 β CDR3

<400> 261

<210> 262

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 65 β signaling peptide

<400> 262

<210> 263

<211> 1830

<212> DNA

<213> Artificial sequence

<220>

<223> Has the full natural sequence of human constant TCR 65

<400> 263

<210> 264

<211> 1806

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of mouse constant TCR 65

<400> 264

<210> 265

<211> 608

<212> PRT

<213> Artificial sequence

<220>

<223> Has the complete sequence of human-specific TCR 65

<400> 265

<210> 266

<211> 600

<212> PRT

<213> Artificial sequence

<220>

<223> Full sequence of TCR 65 with mouse constants

<400> 266

<210> 267

<211> 9

<212> PRT

<213> Human papillomavirus

<220>

<223> E6(18-26) peptide

<400> 267

<210> 268

<211> 10

<212> PRT

<213> Human papillomavirus

<220>

<223> E6(29-38) peptide

<400> 268

<210> 269

<211> 9

<212> PRT

<213> Human papillomavirus

<220>

<223> E6(52-60) peptide

<400> 269

<210> 270

<211> 8

<212> PRT

<213> Human papillomavirus

<220>

<223> E7(86-93) peptide

<400> 270

<210> 271

<211> 9

<212> PRT

<213> Human papillomavirus

<220>

<223> E7(11-19) peptide

<400> 271

<210> 272

<211> 9

<212> PRT

<213> Human papillomavirus

<220>

<223> E7(85-93) peptide

<400> 272

<210> 273

<211> 9

<212> PRT

<213> Human papillomavirus

<220>

<223> E7(82-90) peptide

<400> 273

<210> 274

<211> 9

<212> PRT

<213> Human papillomavirus

<220>

<223> E7(7-15) peptide

<400> 274

<210> 275

<211> 137

<212> PRT

<213> House mouse

<220>

<223> Mouse α homeostasis

<400> 275

<210> 276

<211> 137

<212> PRT

<213> House mouse

<220>

<223> Mouse α homeostasis

<400> 276

<210> 277

<211> 137

<212> PRT

<213> House mouse

<220>

<223> Transmembrane modified/cysteine modified mouse homeostatic α

<400> 277

<210> 278

<211> 136

<212> PRT

<213> House mouse

<220>

<223> Mouse α homeostasis

<400> 278

<210> 279

<211> 137

<212> PRT

<213> House mouse

<220>

<223> Mouse α homeostasis

<400> 279

<210> 280

<211> 137

<212> PRT

<213> House mouse

<220>

<223> Mouse α homeostasis

<220>

<221> VARIANT

<222> (48)...(48)

<223> X=Unknown or other

<220>

<221> VARIANT

<222> (112)...(112)

<223> X=Unknown or other

<220>

<221> VARIANT

<222> (114)...(114)

<223> X=Unknown or other

<220>

<221> VARIANT

<222> (115)...(115)

<223> X=Unknown or other

<400> 280

<210> 281

<211> 137

<212> PRT

<213> House mouse

<220>

<223> Mouse α homeostasis

<400> 281

<210> 282

<211> 137

<212> PRT

<213> House mouse

<220>

<223> Mouse α homeostasis

<400> 282

<210> 283

<211> 172

<212> PRT

<213> House mouse

<220>

<223> Mouse β homeostasis

<400> 283

<210> 284

<211> 173

<212> PRT

<213> House mouse

<220>

<223> Mouse β homeostasis

<400> 284

<210> 285

<211> 173

<212> PRT

<213> House mouse

<220>

<223> Mouse β homeostasis

<220>

<221> VARIANT

<222> (57)...(57)

<223> X=Unknown or other

<400> 285

<210> 286

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence

<400> 286

<210> 287

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Connector

<220>

<221> REPEAT

<222> (5)...(9)

<223> SGGGG repeat 5 or 6 times

<400> 287

<210> 288

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Connector

<400> 288

<210> 289

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> TCR E7(11-19)β total CDR3 common

<220>

<221> VARIANT

<222> (1)...(1)

<223> Xaa=A or S

<220>

<221> VARIANT

<222> (2)...(2)

<223> Xaa=A, I, S or Y

<220>

<221> VARIANT

<222> (3)...(3)

<223> Xaa=S, T or R

<220>

<221> VARIANT

<222> (4)...(4)

<223> Xaa=G, K, R, L, P, S or Q

<220>

<221> VARIANT

<222> (5)...(5)

<223> Xaa=D, G, R, T or W

<220>

<221> VARIANT

<222> (6)...(6)

<223> Xaa=A, E, F, R, S, Q, T or V

<220>

<221> VARIANT

<222> (7)...(7)

<223> Xaa=A, R, G, Y or does not exist

<220>

<221> VARIANT

<222> (8)...(8)

<223> Xaa=G, N, S or does not exist

<220>

<221> VARIANT

<222> (9)...(9)

<223> Xaa=A, D, G, S or Y

<220>

<221> VARIANT

<222> (10)...(10)

<223> Xaa=L, N or Y

<220>

<221> VARIANT

<222> (11)...(11)

<223> Xaa=E or V

<220>

<221> VARIANT

<222> (12)...(12)

<223> Xaa=L or Q

<220>

<221> VARIANT

<222> (13)...(13)

<223> Xaa=F, Y or T

<400> 289

<210> 290

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> TCR E7(11-19)β CDR3 common

<220>

<221> VARIANT

<222> (1)...(1)

<223> Xaa=A or S

<220>

<221> VARIANT

<222> (2)...(2)

<223> Xaa=A or S

<220>

<221> VARIANT

<222> (3)...(3)

<223> Xaa=S or R

<220>

<221> VARIANT

<222> (4)...(4)

<223> Xaa=S, L or P

<220>

<221> VARIANT

<222> (5)...(5)

<223> Xaa=W, D or R

<220>

<221> VARIANT

<222> (6)...(6)

<223> Xaa=R, T or Q

<220>

<221> VARIANT

<222> (7)...(7)

<223> Xaa=R, G, Y or does not exist

<220>

<221> VARIANT

<222> (8)...(8)

<223> Xaa=G, N or S

<220>

<221> VARIANT

<222> (9)...(9)

<223> Xaa=G, D or Y

<220>

<221> VARIANT

<222> (10)...(10)

<223> Xaa=N or L

<400> 290

<210> 291

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> TCR E7(11-19)β CDR3 common

<220>

<221> VARIANT

<222> (1)...(1)

<223> Xaa=A or S

<220>

<221> VARIANT

<222> (2)...(2)

<223> Xaa=I, S or Y

<220>

<221> VARIANT

<222> (3)...(3)

<223> Xaa=S or T

<220>

<221> VARIANT

<222> (4)...(4)

<223> Xaa=G, P, R or T

<220>

<221> VARIANT

<222> (5)...(5)

<223> Xaa=R or T

<220>

<221> VARIANT

<222> (6)...(6)

<223> Xaa=F, S, A or V

<220>

<221> VARIANT

<222> (7)...(7)

<223> Xaa=S or T

<400> 291

<210> 292

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> TCR E7(11-19)α total CDR3 common

<220>

<221> VARIANT

<222> (1)...(1)

<223> Xaa=A or G

<220>

<221> VARIANT

<222> (2)...(2)

<223> Xaa=A, G, P, V or L

<220>

<221> VARIANT

<222> (3)...(3)

<223> Xaa=A, K, N, Y, D or S

<220>

<221> VARIANT

<222> (4)...(4)

<223> Xaa=R, M, I, T or G

<220>

<221> VARIANT

<222> (5)...(5)

<223> Xaa=N, E, L, D, V or does not exist

<220>

<221> VARIANT

<222> (6)...(6)

<223> Xaa=Y, G, N, D or does not exist

<220>

<221> VARIANT

<222> (7)...(7)

<223> Xaa=S, N, G or does not exist

<220>

<221> VARIANT

<222> (8)...(8)

<223> Xaa=G, N, Y or does not exist

<220>

<221> VARIANT

<222> (9)...(9)

<223> Xaa=D, F, G, N or does not exist

<220>

<221> VARIANT

<222> (10)...(10)

<223> Xaa=A, N, Q or Y

<220>

<221> VARIANT

<222> (11)...(11)

<223> Xaa=K, R or N

<220>

<221> VARIANT

<222> (12)...(12)

<223> Xaa=F, L or Y

<220>

<221> VARIANT

<222> (13)...(13)

<223> Xaa=I, M, S, T, V or Y

<400> 292

<210> 293

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> TCR E7(11-19)α CDR3 common

<220>

<221> VARIANT

<222> (4)...(4)

<223> Xaa=M or I

<220>

<221> VARIANT

<222> (5)...(5)

<223> Xaa=E or L

<220>

<221> VARIANT

<222> (6)...(6)

<223> Xaa=G or N

<220>

<221> VARIANT

<222> (7)...(7)

<223> Xaa=S, G or does not exist

<220>

<221> VARIANT

<222> (8)...(8)

<223> Xaa=S or N

<220>

<221> VARIANT

<222> (10)...(10)

<223> Xaa=Y or A

<220>

<221> VARIANT

<222> (11)...(11)

<223> Xaa=K or R

<220>

<221> VARIANT

<222> (13)...(13)

<223> Xaa=T or M

<400> 293

<210> 294

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence

<400> 294

<210> 295

<211> 36

<212> DNA

<213> Homo sapiens

<220>

<223> Spacer (IgG4 hinge)

<400> 295

<210> 296

<211> 119

<212> PRT

<213> Homo sapiens

<220>

<223> Hinge-CH3 spacer

<400> 296

<210> 297

<211> 229

<212> PRT

<213> Homo sapiens

<220>

<223> Hinge-CH2-CH3 spacer

<400> 297

<210> 298

<211> 282

<212> PRT

<213> Homo sapiens

<220>

<223> IgD-hinge-Fc

<400> 298

<210> 299

<211> 357

<212> PRT

<213> Artificial sequence

<220>

<223> tEGFR

<400> 299

<210> 300

<211> 335

<212> PRT

<213> Artificial sequence

<220>

<223> tEGFR

<400> 300

<210> 301

<211> 24

<212> PRT

<213> Artificial sequence

<220>

<223> T2A

<400> 301

<210> 302

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> T2A

<400> 302

<210> 303

<211> 27

<212> PRT

<213> Homo sapiens

<220>

<223> CD28

<400> 303

<210> 304

<211> 66

<212> PRT

<213> Homo sapiens

<220>

<223> CD28

<400> 304

<210> 305

<211> 41

<212> PRT

<213> Homo sapiens

<220>

<223> CD28

<400> 305

<210> 306

<211> 41

<212> PRT

<213> Homo sapiens

<220>

<223> CD28

<400> 306

<210> 307

<211> 42

<212> PRT

<213> Homo sapiens

<220>

<223> 4-1BB

<400> 307

<210> 308

<211> 112

<212> PRT

<213> Homo sapiens

<220>

<223> CD3 ζ

<400> 308

<210> 309

<211> 112

<212> PRT

<213> Homo sapiens

<220>

<223> CD3 ζ

<400> 309

<210> 310

<211> 112

<212> PRT

<213> Homo sapiens

<220>

<223> CD3 ζ

<400> 310

<210> 311

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> P2A

<400> 311

<210> 312

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> F2A

<400> 312

<210> 313

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> E2A

<400> 313

<210> 314

<211> 12

<212> PRT

<213> Homo sapiens

<220>

<223> Separated (IgG4 hinge)

<400> 314

<210> 315

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> P2A

<400> 315

<210> 316

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> P2A

<400> 316

<210> 317

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> P2A

<400> 317

<210> 318

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> P2A

<400> 318

<210> 319

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> P2A

<400> 319

<210> 320

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> P2A

<400> 320

<210> 321

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> P2A

<400> 321

<210> 322

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> P2A

<400> 322

<210> 323

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> P2A

<400> 323

<210> 324

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> P2A

<400> 324

<210> 325

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> P2A

<400> 325

<210> 326

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> P2A

<400> 326

<210> 327

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence

<300>

<308> NCBI NG_001332.3

<309> 2016-08-31

<400> 327

<210> 328

<211> 19

<212> PRT

<213> Homo sapiens

<220>

<223> Signal sequence

<300>

<308> NCBI NG_001333.2

<309> 2016-08-31

<400> 328

<210> 329

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence

<400> 329

<210> 330

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence

<400> 330

<210> 331

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence

<400> 331

<210> 332

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence

<400> 332

<210> 333

<211> 141

<212> PRT

<213> Homo sapiens

<220>

<223> Natural TCR α constant region

<400> 333

<210> 334

<211> 141

<212> PRT

<213> Homo sapiens

<220>

<223> Natural TCR α constant region

<400> 334

<210> 335

<211> 141

<212> PRT

<213> Homo sapiens

<220>

<223> Natural TCR α constant region

<400> 335

<210> 336

<211> 141

<212> PRT

<213> Homo sapiens

<220>

<223> Natural TCR α constant region

<400> 336

<210> 337

<211> 141

<212> PRT

<213> Homo sapiens

<220>

<223> Natural TCR α constant region

<400> 337

<210> 338

<211> 142

<212> PRT

<213> Homo sapiens

<220>

<223> Natural TCR α constant region

<400> 338

<210> 339

<211> 177

<212> PRT

<213> Homo sapiens

<220>

<223> Natural TCR β constant region

<400> 339

<210> 340

<211> 179

<212> PRT

<213> Homo sapiens

<220>

<223> Natural TCR β constant region

<400> 340

<210> 341

<211> 142

<212> PRT

<213> Homo sapiens

<220>

<223> Natural TCR α constant region

<400> 341

<210> 342

<211> 180

<212> PRT

<213> Homo sapiens

<220>

<223> Natural TCR β constant region

<400> 342

<210> 343

<211> 180

<212> PRT

<213> Homo sapiens

<220>

<223> Natural TCR β constant region

<400> 343

<210> 344

<211> 141

<212> PRT

<213> Homo sapiens

<220>

<223> TCR α constant region

<400> 344

<210> 345

<211> 141

<212> PRT

<213> Homo sapiens

<220>

<223> TCR α constant region

<400> 345

<210> 346

<211> 141

<212> PRT

<213> Homo sapiens

<220>

<223> TCR α constant region

<400> 346

<210> 347

<211> 141

<212> PRT

<213> Homo sapiens

<220>

<223> TCR α constant region

<400> 347

<210> 348

<211> 141

<212> PRT

<213> Homo sapiens

<220>

<223> TCR α constant region

<400> 348

<210> 349

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence

<400> 349

<210> 350

<211> 179

<212> PRT

<213> Homo sapiens

<220>

<223> TCR β constant region

<400> 350

<210> 351

<211> 180

<212> PRT

<213> Homo sapiens

<220>

<223> TCR β constant region

<400> 351

<210> 352

<211> 180

<212> PRT

<213> Homo sapiens

<220>

<223> TCR β constant region

<400> 352

<210> 353

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence

<400> 353

<210> 354

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence

<400> 354

<210> 355

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Signal sequence

<400> 355

<210> 356

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> TCR E7(11-19)α CDR3 common

<220>

<221> VARIANT

<222> (1)...(1)

<223> Xaa=A or G

<220>

<221> VARIANT

<222> (2)...(2)

<223> Xaa=A, G, P, V or L

<220>

<221> VARIANT

<222> (3)...(3)

<223> Xaa=A, K, N, Y, D or S

<220>

<221> VARIANT

<222> (4)...(4)

<223> Xaa=R, M, I, T or G

<220>

<221> VARIANT

<222> (5)...(5)

<223> Xaa=N, E, L, V or does not exist

<220>

<221> VARIANT

<222> (6)...(6)

<223> Xaa=Y, G, N, D or does not exist

<220>

<221> VARIANT

<222> (7)...(7)

<223> Xaa=S, N, G or does not exist

<220>

<221> VARIANT

<222> (8)...(8)

<223> Xaa=G, N, Y or does not exist

<220>

<221> VARIANT

<222> (9)...(9)

<223> Xaa=D, F, G, N or does not exist

<220>

<221> VARIANT

<222> (10)...(10)

<223> Xaa=A, N, Q or Y

<220>

<221> VARIANT

<222> (11)...(11)

<223> Xaa=K, R or N

<220>

<221> VARIANT

<222> (12)...(12)

<223> Xaa=F, L or Y

<220>

<221> VARIANT

<222> (13)...(13)

<223> Xaa=I, M, S, T, V or Y

<400> 356

<210> 357

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> TCR E7(11-19)β CDR3 common

<220>

<221> VARIANT

<222> (1)...(1)

<223> Xaa=A or S

<220>

<221> VARIANT

<222> (2)...(2)

<223> Xaa=A, I, S or Y

<220>

<221> VARIANT

<222> (3)...(3)

<223> Xaa=S, T or R

<220>

<221> VARIANT

<222> (4)...(4)

<223> Xaa=G, K, R, L, P, S or Q

<220>

<221> VARIANT

<222> (5)...(5)

<223> Xaa=D, G, T or W

<220>

<221> VARIANT

<222> (6)...(6)

<223> Xaa=A, E, F, R, S, Q, T or V

<220>

<221> VARIANT

<222> (7)...(7)

<223> Xaa=A, R, G, Y or does not exist

<220>

<221> VARIANT

<222> (8)...(8)

<223> Xaa=G, N, S or does not exist

<220>

<221> VARIANT

<222> (9)...(9)

<223> Xaa=A, D, G, S or Y

<220>

<221> VARIANT

<222> (10)...(10)

<223> Xaa=L, N or Y

<220>

<221> VARIANT

<222> (11)...(11)

<223> Xaa=E or V

<220>

<221> VARIANT

<222> (12)...(12)

<223> Xaa=L or Q

<220>

<221> VARIANT

<222> (13)...(13)

<223> Xaa=F, Y or T

<400> 357

<210> 358

<211> 537

<212> DNA

<213> Homo sapiens

<220>

<223> TCR β constant region

<400> 358

<210> 359

<211> 295

<212> PRT

<213> Artificial sequence

<220>

<223> β with human constant sequence

<400> 359

<210> 360

<211> 309

<212> PRT

<213> Artificial sequence

<220>

<223> β with human constant sequence and signal peptide

<400> 360

<210> 361

<211> 927

<212> DNA

<213> Artificial sequence

<220>

<223> β with human constant sequence and signal peptide

<400> 361

<210> 362

<211> 600

<212> PRT

<213> Artificial sequence

<220>

<223> Has the complete sequence of human constant TCR

<400> 362

<210> 363

<211> 1800

<212> DNA

<213> Artificial sequence

<220>

<223> Has the complete sequence of human constant TCR

<400> 363

<210> 364

<211> 1893

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 56 (nt)

<400> 364

<210> 365

<211> 1803

<212> DNA

<213> Artificial sequence

<220>

<223> Full sequence of human constant TCR 57 (nt)

<400> 365

<210> 366

<211> 407

<212> DNA

<213> Artificial sequence

<220>

<223> TCR α constant region, Homo sapiens (nt)

<400> 366

<210> 367

<211> 407

<212> DNA

<213> Artificial sequence

<220>

<223> TCR α constant region, Homo sapiens (nt)

<400> 367

<210> 368

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> P2A artificial (nt)

<400> 368

<210> 369

<211> 296

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 56 β (aa) with human constant sequence

<400> 369

<210> 370

<211> 292

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 58 β (aa) with human constant sequence

<400> 370

<210> 371

<211> 292

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 59 β (aa) with human constant sequence

<400> 371

<210> 372

<211> 292

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 60 β (aa) with human constant sequence

<400> 372

<210> 373

<211> 292

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 61 β (aa) with human constant sequence

<400> 373

<210> 374

<211> 296

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 62 β (aa) with human constant sequence

<400> 374

<210> 375

<211> 295

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 63 β (aa) with human constant sequence

<400> 375

<210> 376

<211> 293

<212> PRT

<213> Artificial sequence

<220>

<223> TCR with human constant sequence 64 β(aa)

<400> 376

<210> 377

<211> 294

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 65 β (aa) with human constant sequence

<400> 377

<210> 378

<211> 320

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 56 β(aa) with human constant sequence and signal peptide

<400> 378

<210> 379

<211> 311

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 58 β(aa) with human constant sequence and signal peptide

<400> 379

<210> 380

<211> 311

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 59 β(aa) with human constant sequence and signal peptide

<400> 380

<210> 381

<211> 311

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 60 β(aa) with human constant sequence and signal peptide

<400> 381

<210> 382

<211> 311

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 61 β(aa) with human constant sequence and signal peptide

<400> 382

<210> 383

<211> 315

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 62 β(aa) with human constant sequence and signal peptide

<400> 383

<210> 384

<211> 314

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 63 β(aa) with human constant sequence and signal peptide

<400> 384

<210> 385

<211> 312

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 64 β(aa) with human constant sequence and signal peptide

<400> 385

<210> 386

<211> 313

<212> PRT

<213> Artificial sequence

<220>

<223> TCR 65 β(aa) with human constant sequence and signal peptide

<400> 386

<210> 387

<211> 1890

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 56 (nt)

<400> 387

<210> 388

<211> 1899

<212> DNA

<213> Artificial sequence

<220>

<223> Full sequence of TCR 56 codons with human constants (nt)

<400> 388

<210> 389

<211> 1797

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 57 (nt)

<400> 389

<210> 390

<211> 1797

<212> DNA

<213> Artificial sequence

<220>

<223> Full sequence of TCR 57 codons optimized with human constants (nt)

<400> 390

<210> 391

<211> 1818

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 58 (nt)

<400> 391

<210> 392

<211> 1824

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 59 (nt)

<400> 392

<210> 393

<211> 1830

<212> DNA

<213> Artificial sequence

<220>

<223> Has the natural complete sequence of human constant TCR 60 (nt)

<400> 393

<210> 394

<211> 1821

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 61 (nt)

<400> 394

<210> 395

<211> 1824

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of 62 constant human TCR (nt)

<400> 395

<210> 396

<211> 1818

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 63 (nt)

<400> 396

<210> 397

<211> 1836

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of 64 TCRs with constant human characteristics (nt)

<400> 397

<210> 398

<211> 1827

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 65 (nt)

<400> 398

<210> 399

<211> 1902

<212> DNA

<213> Artificial sequence

<220>

<223> Full sequence of TCR 56 codons with human constants (nt)

<400> 399

<210> 400

<211> 1896

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 56 (nt)

<400> 400

<210> 401

<211> 1905

<212> DNA

<213> Artificial sequence

<220>

<223> Full sequence of TCR 56 codons with human constants (nt)

<400> 401

<210> 402

<211> 1803

<212> DNA

<213> Artificial sequence

<220>

<223> Optimized full sequence with constant human TCR 57 encoding (nt)

<400> 402

<210> 403

<211> 1824

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 58 (nt)

<400> 403

<210> 404

<211> 1824

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 59 (nt)

<400> 404

<210> 405

<211> 1836

<212> DNA

<213> Artificial sequence

<220>

<223> Has the natural complete sequence of human constant TCR 60 (nt)

<400> 405

<210> 406

<211> 1827

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 61 (nt)

<400> 406

<210> 407

<211> 1830

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of 62 constant human TCR (nt)

<400> 407

<210> 408

<211> 1824

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 63 (nt)

<400> 408

<210> 409

<211> 1842

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of 64 TCRs with constant human characteristics (nt)

<400> 409

<210> 410

<211> 1833

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 65 (nt)

<400> 410

<210> 411

<211> 960

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 56 (nt)

<400> 411

<210> 412

<211> 969

<212> DNA

<213> Artificial sequence

<220>

<223> Full sequence of TCR 56 codons with human constants (nt)

<400> 412

<210> 413

<211> 927

<212> DNA

<213> Artificial sequence

<220>

<223> Full sequence of TCR 57 codons optimized with human constants (nt)

<400> 413

<210> 414

<211> 933

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 58 (nt)

<400> 414

<210> 415

<211> 933

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 59 (nt)

<400> 415

<210> 416

<211> 933

<212> DNA

<213> Artificial sequence

<220>

<223> Has the natural complete sequence of human constant TCR 60 (nt)

<400> 416

<210> 417

<211> 933

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 61 (nt)

<400> 417

<210> 418

<211> 945

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of 62 constant human TCR (nt)

<400> 418

<210> 419

<211> 942

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 63 (nt)

<400> 419

<210> 420

<211> 870

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized TCR 56 α (nt) with codon-optimized human constant

<400> 420

<210> 421

<211> 858

<212> DNA

<213> Artificial sequence

<220>

<223> Codon-optimized TCR 56 α (nt) using codon-optimized mouse constants

<400> 421

<210> 422

<211> 1878

<212> DNA

<213> Artificial sequence

<220>

<223> Full sequence of mouse constant TCR 56 codons optimized (nt)

<400> 422

<210> 423

<211> 936

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of 64 TCRs with constant human characteristics (nt)

<400> 423

<210> 424

<211> 939

<212> DNA

<213> Artificial sequence

<220>

<223> Full natural sequence of human constant TCR 65 (nt)

<400> 424

Claims (28)

An anti-HPV 16 E7 T cell receptor (TCR) or an antigen-binding fragment thereof, comprising an α chain containing a variable α (Vα) region and a β chain containing a variable β (Vβ) region, wherein: the Vα region comprises a complementary determining region 1 (CDR-1), a complementary determining region 2 (CDR-2) and a complementary determining region 3 (CDR-3), which respectively comprise the amino acid sequences of SEQ ID NOs: 48, 49 and 50, and the Vβ region comprises CDR-1, CDR-2 and CDR-3, which respectively comprise the amino acid sequences of SEQ ID NOs: 61, 62 and 63. The T cell receptor (TCR) or antigen-binding fragment thereof of claim 1, wherein: the Vα region comprises the amino acid sequence shown in SEQ ID NO: 47 or an amino acid sequence having at least 90% sequence identity therewith, and the Vβ region comprises the amino acid sequence shown in SEQ ID NO: 60 or an amino acid sequence having at least 90% sequence identity therewith. As claimed in claim 1, the TCR or antigen-binding fragment thereof binds to or recognizes a peptide antigenic determinant of human papillomavirus (HPV) 16 E7 in the context of an MHC molecule, and the peptide antigenic determinant is or comprises E7(11-19)YMLDLQPET (SEQ ID NO: 271). The TCR or antigen-binding fragment thereof of claim 1, wherein the Vα region and the Vβ region comprise the amino acid sequences of SEQ ID NOs: 47 and 60, respectively. As claimed in claim 1, the TCR or antigen-binding fragment thereof, wherein the α chain further comprises an α constant (Cα) region and/or the β chain further comprises a β constant (Cβ) region. As in claim 5, the TCR or its antigen-binding fragment, wherein the Cα region and the Cβ region are mouse constant regions. As in claim 5, the TCR or antigen-binding fragment thereof, wherein the Cα region and the Cβ region are human constant regions. The TCR or antigen-binding fragment thereof of claim 5, wherein the Cα region and/or the Cβ region comprises one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the α chain and the β chain. The TCR or antigen-binding fragment thereof of claim 5, wherein the α chain comprises the amino acid sequence shown in SEQ ID NO: 43 or an amino acid sequence having at least 90% sequence identity therewith; and/or the β chain comprises the amino acid sequence shown in SEQ ID NO: 359 or an amino acid sequence having at least 90% sequence identity therewith. The TCR or antigen-binding fragment thereof of claim 5, wherein: the Cα region comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 90% sequence identity therewith; and/or the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 350 or an amino acid sequence having at least 90% sequence identity therewith. The TCR or antigen-binding fragment thereof of claim 5, wherein: the Cα region comprises the amino acid sequence shown in SEQ ID NO: 14; and the Cβ region comprises the amino acid sequence shown in SEQ ID NO: 350. The TCR or antigen-binding fragment thereof as claimed in claim 5, wherein the TCR is encoded by a polynucleotide encoding the sequence shown in SEQ ID NO: 362. The TCR or antigen-binding fragment thereof as claimed in claim 1 is human. A TCR or antigen-binding fragment thereof as claimed in claim 1, wherein the antigen specificity is at least partially CD8-independent. A nucleic acid molecule encoding a TCR or an antigen-binding fragment thereof or an α chain or β chain thereof as described in any one of claims 1 to 14. A nucleic acid molecule as claimed in claim 15, wherein the nucleic acid molecule encodes a polypeptide comprising the sequence shown in SEQ ID NO: 362 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. A vector comprising the nucleic acid of claim 15 or 16. An engineered cell comprising a TCR or antigen-binding fragment thereof as described in any one of claims 1 to 14, a nucleic acid as described in claim 15 or 16, or a vector as described in claim 17. The engineered cell of claim 18, wherein the engineered cell is a T cell. An engineered cell as claimed in claim 18, comprising gene disruption of the T cell receptor α homeostatic ( TRAC ) gene and/or the T cell receptor β homeostatic ( TRBC ) gene. An engineered cell as claimed in claim 20, wherein the gene disruption comprises disruption of the T cell receptor alpha homeostasis ( TRAC ) gene. A method for producing an engineered cell as in any one of claims 18 to 21, comprising introducing a vector as in claim 17 into a cell in vitro or ex vivo. A pharmaceutical composition comprising an engineered cell as described in any one of claims 18 to 21. The pharmaceutical composition of claim 23, wherein the engineered cells comprise CD4+ and/or CD8+ T cells. The pharmaceutical composition of claim 23, wherein the engineered cells include CD4+ and CD8+ T cells. The pharmaceutical composition of claim 23 further comprises a pharmaceutically acceptable excipient. A use of a pharmaceutical composition as claimed in any one of claims 23 to 26 for the manufacture of a medicament for treating a disease or condition associated with HPV in an individual. For use as claimed in claim 27, wherein the disease or condition is cancer.